Compositions and methods related to graft versus host disease and treatments thereof

ABSTRACT

Embodiments of the present invention illustrate methods of treating and preventing transplantation and side effects associated with transplantation. In particular, the present invention relates to compositions and methods for inhibition of graft rejection and promotion of graft survival. Thus, the invention relates to modulation of cellular activities, including graft rejection, promotion of graft survival, graft versus host rejection and conditions commonly associated with graft rejection. More particularly, the present invention relates to the inhibitory compounds comprising naturally occurring and man-made inhibitors of serine protease and inducers of other alpha1-antitrypsin activities.

PRIORITY

This application is a continuation of U.S. application Ser. No.11/916,521 filed Dec. 4, 2007, which is a national stage application ofPCT Application No. PCT/US2006/22436, filed Jun. 7, 2006, which claimspriority to U.S. Provisional Application No. 60/687,580 filed Jun. 7,2005. All prior applications are incorporated herein in their entiretyby reference for all purposes.

FIELD

Embodiments of the present invention relate to compositions and methodsfor treatment of subjects in need of or having a transplant. Inparticular, embodiments of the present invention relate to compositionsand methods for treatment of conditions associated with transplantationsin a subject, for example, graft rejection. More particularly, thepresent invention relates to compositions and uses of alpha1-antitrypsin(α1-antitrypsin) and agents with α1-antitrypsin-like activity and/orcompositions and uses of serine protease inhibitors.

BACKGROUND Serine Proteases

Serine proteases serve an important role in human physiology bymediating the activation of vital functions. In addition to their normalphysiological function, serine proteases have been implicated in anumber of pathological conditions in humans. Serine proteases arecharacterized by a catalytic triad consisting of aspartic acid,histidine and serine at the active site.

Naturally occurring serine protease inhibitors have been classified intofamilies primarily on the basis of the disulfide bonding pattern and thesequence homology of the reactive site. Serine protease inhibitors,including the group known as serpins, have been found in microbes, inthe tissues and fluids of plants, animals, insects and other organisms.At least nine separate, well-characterized proteins are now identified,which share the ability to inhibit the activity of various proteases.Several of the inhibitors have been grouped together, namelyα1-antitrypsin-proteinase inhibitor, secretory leukocyte proteaseinhibitor or SLPI, antithrombin III, antichymotrypsin, C1-inhibitor, andα2-antiplasmin, which are directed against various serine proteases,i.e., leukocyte elastase, thrombin, cathepsin G, chymotrypsin,plasminogen activators, and plasmin. These inhibitors are members of theα1-antitrypsin-proteinase inhibitor class. The protein α2-macroglobulininhibits members of all four classes of endogenous proteases: serine,cysteine, aspartic, and metalloproteases. However, other types ofprotease inhibitors are class specific. For example, theα1-antitrypsin-proteinase inhibitor (also known as (α1-antitrypsin orAAT) and inter-alpha-trypsin inhibitor inhibit only serine proteases,α1-cysteine protease inhibitor inhibits cysteine proteases, andα1-anticollagenase inhibits collagenolytic enzymes of the metalloenzymeclass.

The normal plasma concentration of ATT ranges from 1.3 to 3.5 mg/mlalthough it can behave as an acute phase reactant and increase 3-4-foldduring host response to inflammation and/or tissue injury such as withpregnancy, acute infection, and tumors. It easily diffuses into tissuespaces and forms a 1:1 complex with target proteases, principallyneutrophil elastase. Other enzymes such as trypsin, chymotrypsin,cathepsin G, plasmin, thrombin, tissue kallikrein, and factor Xa canalso serve as substrates. The enzyme/inhibitor complex is then removedfrom circulation by binding to serpin-enzyme complex (SEC) receptor andcatabolized by the liver and spleen. ATT appears to represent animportant part of the defense mechanism against activity by serineproteases.

α1-antitrypsin is one of few naturally occurring mammalian serineprotease inhibitors currently approved for the clinical therapy ofprotease imbalance. Therapeutic α1-antitrypsin has been commerciallyavailable since the mid 1980's and is prepared by various purificationmethods (see for example Bollen et al., U.S. Pat. No. 4,629,567;Thompson et al., U.S. Pat. Nos. 4,760,130; 5,616,693; WO 98/56821).Prolastin is a trademark for a purified variant of α1-antitrypsin and iscurrently sold by Talectris Company (U.S. Pat. No. 5,610,285 Lebing etal., Mar. 11, 1997). Recombinant unmodified and mutant variants ofα1-antitrypsin produced by genetic engineering methods are also known(U.S. Pat. No. 4,711,848); methods of use are also known, e.g.,(α1-antitrypsin gene therapy/delivery (U.S. Pat. No. 5,399,346).

Graft Rejection

There are many diseases that culminate in organ dysfunction or failure.Representative non-limiting examples include renal failure due todiabetes melitus, hypertension, urinary output obstruction, drug-inducedtoxicity, or hypoperfusion, as well as cardiac dysfunction due toischemic coronary artery disease, cardiomyopathy/infection, orvalvulopathy. Pulmonary diseases include substantial damage due tochronic obstructive pulmonary disease (COPD, including chronicbronchitis and emphysema), AAT deficiency, cystic fibrosis, andinterstitial fibrosis. Under certain conditions, the only therapeuticoption for treatment of a subject may be organ transplantation.Pancreatic-islet transplantation provides diabetic patients with theonly option for a tightly-controlled blood glucose level, as proven tobe essential for prevention of diabetic complications. In the case ofislets, post-transplant inflammation, which precedes immune rejection,is a critical determinant of graft survival. This early inflammation ismediated by cells other than the impending allospecific immune cells.

One challenge to therapeutic transplantation is the damaging effects ofthe host immune system on the transplant. MHC molecules exist on thesurfaces of cells and the particular structures of MHC molecules aretypically unique for each individual (with the exception of identicaltwins, where the MHC molecule complements are identical). The immunesystem is programmed to attack foreign or “non-self” MHC-bearingtissues. For these reasons, when an organ or tissue is transplanted intoa recipient, an effort is made to optimize the degree of tissue matchingbetween donor and recipient. MHC antigens are characterized for therecipient and donors. Matching a donor to an allograft recipient by MHCstructure reduces the magnitude of the rejection response. An archetypalexample is blood group matching. Most transplants are allografts thatoccur between non-identical members of the same species. Since thesematches are imperfect, there is an expected graft rejection immuneresponse associated with allografts. Current methods used, in order toenhance graft survival, include medications to suppress the immuneresponse which can result in graft rejection. These medications arereferred to immunosuppressant or antirejection drugs, such asprednisone, cyclosporine A, and cyclophosphamide, to name a few. Asmentioned above, local inflammation is experienced immediately aftergrafting, and cells that are particularly sensitive to non-specificinflammation, such as islets, can endure graft dysfunction more severelythan other types.

Despite advances in the field of antirejection therapy, graftmaintenance remains a challenge since the available antirejectiontherapies are imperfect. For example, immunosuppression enhances therisk for opportunistic infection or neoplasia. Toxicities abound andinclude, but are not limited to, diabetes, organ dysfunction, renalfailure, hepatic dysfunction, hematological defects, neuromuscular andpsychiatric side effects, and many others. Therefore, there is a needfor a more effective anti-rejection medical treatment that prolong graftsurvival and improve the quality of life.

Bone marrow transplantation is a unique kind of transplant where immunecells from a donor are transferred into a recipient, thereby conferringthe donor immune system into the recipient. Here, the graft is capableof generating an immune response against the host, and this is termed“graft versus host” disease (GVHD). Immunosuppressive and antimicrobialtreatment is required to block adverse consequences of GVHD, and a needexists for safer and more effective inhibitors of the adverse effects bythe graft.

Because of some of the difficulties and inadequacies of conventionaltherapy for treating transplantation complications and associatedside-effects, new therapeutic modalities are needed.

SUMMARY

Embodiments of the present invention provide for methods for treating asubject having or in need of a transplant. In accordance with theseembodiments, a subject may be treated with a composition for reducingthe risk of a transplant rejection or a side-effect of a transplantrejection in a subject. In accordance with this method the subject canbe administered a composition including a compound that is capable ofsignificantly reducing serine protease activity. The composition may beadministered before transplantation, during transplantation, aftertransplantation or combination thereof. In addition, the composition mayfurther include one or more anti-transplant rejection agent,anti-inflammatory agent, immunosuppressive agent, immunomodulatoryagent, anti-microbial agent, or a combination thereof.

In certain embodiments of the invention, a composition capable ofsignificantly reducing serine protease activity can includealpha-1-antitrypsin, an analog thereof or a combination thereof Atransplant of the present invention may include an organ transplantand/or a non-organ transplant. For example lung, kidney, heart, liver,cornea, skin, stem cells, soft tissue (e.g. facial componenttransplant), intestinal transplants, bone marrow, pancreatic islet,pancreas transplant or combination thereof are contemplated.

Embodiments of the present invention provide for methods forameliorating symptoms or signs experienced by a subject having or inneed of a transplant. In accordance with these embodiments, symptoms orsigns may include conditions associated with graft versus host disease(GVHD), or graft rejection. In one example, methods disclosed herein maybe used to treat a subject undergoing bone marrow transplantation. Inanother embodiment, symptoms or signs may include but is not limited toone or more of the following, kidney failure, lung failure, heartfailure, malaise, fever, dry cough, anorexia, weight loss, myalgias, andchest pains, ventilatory compromise, sweating, nausea, vomiting, fever,abdominal pain, bloody diarrhea, mucosal ulcerations, reduced renalfunction (increased creatinine, decreased urine output), reducedpulmonary function (increased shortness of breadth, fever, cough,sputum, hypoxemia), reduced cardiac function (shortness of breach, chestpain, fatigue, pulmonary or peripheral edema, valvulopathy), reducedislet function (increased glucose, diabetes melitus), graft versus hostdisease (gastrointestinal (GI) ulceration, pulmonary failure, skinulceration, coagulopothy, CNS dysfunction (mental status changes, coma)CMV (cytomeglovirus infection, viral, fungal parasitic infection)).

Embodiments of the present invention provide methods for promotingprolonged graft survival and function in a subject includingadministering to a subject in need thereof a therapeutically effectiveamount of a composition including a substance exhibiting al-antitrypsinor α1-antitrypsin analog or inhibitor of serine protease activity or afunctional derivative thereof.

Embodiments of the present invention provide for methods for treating asubject in need of an immunotolerance therapy. In accordance with theseembodiments, a subject may be treated with a composition for reducingthe risk of a dysfunctional immune responses or a side-effect of adysfunctional immune response in a subject. In another embodiment,methods herein provide for inducing immune tolerance specific for agraft and/or reduce the need for immunosuppressive therapy. Inaccordance with this embodiment, the immune system of the transplantrecipient may have reduced or lost the specific ability to attack thegraft while maintaining its ability to mount any other type of immuneattack. In accordance with this method the subject can be administered acomposition including a compound that is capable of significantlyreducing serine protease activity or other activity associated withα1-antitrypsin or α1-antitrypsin analog. In certain embodiments, acomposition capable of significantly reducing serine protease activitycan include alpha-1-antitrypsin, an analog thereof or a combinationthereof. In accordance with these embodiments, one example forimmunotolerance therapy can include inhibiting cytokine production.

Embodiments of the present invention provide for methods for reducingTNFα (tumor necrosis factor alpha) levels in a subject includingadministering a composition including alpha-1-antitrypsin, an analogthereof or a combination thereof to a subject in need of such atreatment.

Embodiments of the present invention provide for methods for treating asubject in need of an immunotolerance therapy. In accordance with theseembodiments methods are provided for reducing NO production and/orreducing apoptosis and/or inhibiting cytomegleovirus (infection andreactivation) including administering a composition including a compoundthat is capable of significantly reducing serine protease activityand/or other alpha-1-antitrypsin activity. In certain embodiments of theinvention, a composition capable of significantly reducing serineprotease activity and/or mimicking other alpha-1-antitrypsin activitycan include alpha-1-antitrypsin, an analog thereof, or a combinationthereof.

In certain embodiments of the present invention, the anti-inflammatorycompound or immunomodulatory drug can include but is not limited to oneor more of interferon, interferon derivatives including betaseron,beta-interferon, prostane derivatives including iloprost, cicaprost;glucocorticoids including cortisol, prednisolone, methyl-prednisolone,dexamethasone; immunsuppressives including cyclosporine A, FK-506,methoxsalene, thalidomide, sulfasalazine, azathioprine, methotrexate;lipoxygenase inhibitors comprising zileutone, MK-886, WY-50295,SC-45662, SC-41661A, BI-L-357; leukotriene antagonists; peptidederivatives including ACTH and analogs thereof; soluble TNF-receptors;TNF-antibodies; soluble receptors of interleukins, other cytokines,T-cell-proteins; antibodies against receptors of interleukins, othercytokines, T-cell-proteins; and calcipotriols; Celcept®, mycophenolatemofetil, and analogues thereof taken either alone or in combination.

Embodiments of the present invention provide for methods for reducinggraft rejection in a subject. In accordance with these embodiments, asubject may be treated with a composition for reducing the risk of graftrejection responses or a side-effect of a graft rejection response in asubject. In accordance with this method, the subject can be administereda composition including a compound that is capable of significantlyreducing serine protease activity. In certain embodiments, a compositioncapable of significantly reducing serine protease activity can includeα1-antitrypsin, an analog thereof or a combination thereof. In oneexample, reducing graft rejection may include reducing the symptomsassociated with graft rejection in a subject having an organ transplant,such as a kidney transplant or a bowel transplant or a non-organtransplant, such as a bone marrow transplant soft tissue transplant.

In yet another embodiment, the present invention may include combinationtherapies including compositions exhibiting α1-antitrypsin, an analogthereof, or substance with serine protease inhibitor activity. Forexample, a composition may include α1-antitrypsin and another serineprotease inhibitor administered simultaneously or in separatecompositions.

In accordance with embodiments disclosed herein, any of the disclosedcompositions may be used to ameliorate symptoms associated with atransplant. These symptoms may include but are not limited to,infiltration of graft with cells and/or serum factors (for example,complement, anti-graft antibodies), increased cytokine and/or chemokineproduction, increased nitric oxide production, increased apoptosis andcell death, and increased immune response against the transplant tissueand/or cells.

In another aspect, the present invention provides for a method ofameliorating a symptom or sign associated with transplantation in asubject in need of said amelioration. In accordance with thisembodiment, a composition may be administered to a subject such as apharmaceutically effective amount of a substance of α1-antitrypsin, ananalog thereof or serine protease inhibitor activity, wherein thecomposition is capable of reducing, preventing or inhibiting serineprotease or protease activity and/or binds to the sec receptor or otheractivity.

In certain embodiments, synthetic and/or naturally occurring peptidesmay be used in compositions and methods of the present invention forexample, providing serine protease inhibitor activity. Homologues,natural peptides, with sequence homologies to AAT including peptidesdirectly derived from cleavage of AAT may be used or other peptides suchas, peptides that inhibit serine proteases or have AAT-like activity.Other peptidyl derivatives, e.g., aldehyde or ketone derivatives of suchpeptides are also contemplated herein. Without limiting to AAT andpeptide derivatives of AAT, compounds like oxadiazole, thiadiazole andtriazole peptoids and substances comprising certain phenylenedialkanoateesters, CE-2072, UT-77, and triazole peptoids may be used. Examples ofanalogues are TLCK (tosyl-L-lysine chloromethyl ketone) or TPCK(tosyl-L-phenylalanine chloromethyl ketone).

In other embodiments, an agent that reduces the occurrence of graftrejection, promotes prolonged graft function or promotes prolongedallograft survival can also be an inhibitor of serine protease activity,an inhibitor of elastase, or an inhibitor of proteinase-3. An inhibitorof serine protease activity can include, but is not limited to, smallorganic molecules including naturally-occurring, synthetic, andbiosynthetic molecules, small inorganic molecules includingnaturally-occurring and synthetic molecules, natural products includingthose produced by plants and fungi, peptides, variants ofα1-antitrypsin, chemically modified peptides, and proteins.

In some embodiments, AAT peptides contemplated for use in thecompositions and methods of the present invention are also intended toinclude any and all of those specific AAT peptides other than the 10amino acid AAT peptides of SEQ ID NO. 1 depicted supra. Any combinationof consecutive amino acids depicting a portion of AAT or AAT-likeactivity may be used, such as amino acids 2-12, amino acids 3-13, 4-14,etc. of SEQ ID NO. 1, as well as any and all AAT peptide fragmentscorresponding to select amino acids of SEQ ID NO. 1. Applicants areherein entitled to compositions based upon any and all AAT peptidevariants based upon the amino acid sequence depicted in SEQ ID NO. 1.

In one aspect of the invention, the pharmaceutical compositions of thepresent invention are administered orally, systemically, via an implant,intravenously, topically, intrathecally, intratracheally,intracranially, subcutaneously, intravaginally, intraventricularly,intranasally such as inhalation, mixed with grafts by flushing of organor suspension of cells, or any combination thereof.

As such, those skilled in the art will appreciate that the conception,upon which this disclosure is based, can readily be used as a basis fordesigning other methods for carrying out the several features andadvantages of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain embodiments of the presentinvention. The embodiments may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof specific embodiments presented herein.

FIG. 1A-1D illustrates an exemplary method of treating islet allograftswith AAT. Islets from DBA/2 mice (H-2d) were transplanted under therenal capsule of streptozotocin-induced hyperglycemic C57BL/6 mice(H-2b). (A) Glucose levels from days 6-18. (B) Treatment protocols.Control and full AAT treatment are described in panel A. Early AATtreatment consists of treatment on days −1, 1 and 3 (2 mg, n=3). LateAAT treatment consists of treatment from day 2 and on every 2 days (2mg, n=3). (C) Effect of mouse anti-human-AAT antibodies. Dashed lineindicates post transplantation glucose levels of a mouse under full AATtreatment protocol (see A, B) that was immunized by multipleadministrations of human AAT prior to transplantation (1 representative,n=3). Solid line indicates glucose levels of a non-immunized mousetreated under full AAT treatment protocol (1 representative, n=10).Arrow indicates detection of treatment-induced, anti-human-AATantibodies in the non-immunized representative mouse. (D) Comparison ofday 15 post-transplantation glucose levels in mice that were under fulltreatment protocol with ALB (n=3) or AAT (non-immunized n=10, immunizedn=3). Of the AAT-treated group, antibodies were detected on day 15 in3/3 immunized mice and in 6/10 non-immunized mice.

FIG. 2A-2D illustrates an exemplary method of the effect of AAT onthioglycolate-elicited peritoneal cellular infiltrates. (A) Total cellpopulation of lavaged cells of (o) saline or ( ) AAT-treated (5^(Δ) mg)thioglycolate-injected mice. (B) Percent cell population fromsaline-treated mice at 48 hours. (C) Oxidation of AAT. (D)Identification of elicited macrophages and neutrophils.

FIG. 3A-3C illustrates an exemplary method of the effect of AAT onMHC-incompatible, NIH-3T3-fibroblast-elicited peritoneal cellularinfiltrates. (A) Cell numbers. The number of cells in each subpopulationwas calculated from the percentages obtained by FACS analysis, and totalnumber of cells in the infiltrate. (B) Representative FACS analysis. (C)Effect of AAT on intensity and function of infiltrate elicited by isletallograft. Left, Hematoxilyn and Eosin (H&E) staining of day 7 isletallografts. Right, Immunohistochemistry (IHC) with anti-insulinantibodies of day 15 islet grafts. R, renal parenchyma, G, graft, C,renal capsule.

FIG. 4A-4H illustrates an exemplary method of the effect of AAT on isletresponses. (A-D) Mean±SEM of A. nitric levels, B. Cell viability and C.MIP-1α levels. Dashed line represents islets incubated at one-30th theconcentration of IFNγ/IL-1β. D. TNFα levels. (E) Insulin inductionassay. (F) Streptozotocin toxicity. Each image depicts a representativeislet from one pancreas. (G) Cellular content of islets. (H) MHC classII expression.

FIG. 5A-5D illustrates the effect of AAT on TNFα. (A) Islets fromC57BL/6 mice were cultured (100 islets/well in triplicate) in thepresence of AAT (0.5 mg/ml) or TACE inhibitor (10 mM) 1 hour beforestimulation by IFNγ (5 ng/ml) plus IL-1β (10 ng/ml). Left, mean±SEMchange in TNFα in supernatants after 72 hours of incubation. Right,mean±SEM fold change in membrane TNFα on islet cells after 5 hours ofincubation, according to FACS analysis. (B) Representative FACS analysisof membrane TNFα on stimulated islet cells in the absence (open area) orpresence (shaded area) of AAT. (C) Streptozotocin-induced hyperglycemia.

FIG. 6A-6D illustrates the effect of AAT on Islet allografttransplantation. 6A illustrates the time course study aftertransplantation. 6B illustrates an immune infiltrate found outside thegraft area. 6C illustrates an increase in the presence of CD4+ and acomparative decrease in monocytes and neutrophils. 6D illustrates levelsof glucose reflecting a level of tolerance with respect to daysfollowing allografting of the same donor (left) and a 3^(rd) donorre-graft (right), indicating induction of specific immune tolerance.

FIG. 7A-7E illustrates the production of AAT by islet cell andreflection of islet graft survival. 7A illustrates a time courseexpression of mouse AAT mRNA after cytokine production (IL-1β and IFNγ)(left) and at 8 hours (right). 7B illustrates an example of islet injuryduring pancreatitis; the histology of normal islets (top left), thehistology of islets of an inflamed pancreas (top right) and expressionof mouse AAT in islets obtained from the pancreata in an acutepancreatitis model (bottom). 7C illustrates an example of samples ofislet allografts taken post grafting and the percent change in AAT mRNAlevels were assessed. 7D illustrates an example of islet protection fromcytokine injury with endogenous AAT by introducing oncostatin M (aninterleukin 6 (IL-6) family member) that induces AAT expression inislets, oncostatin M and AAT levels (top left); nitric oxide andviability levels assessed (top right) and nitric oxide productionrepresenting islet viability after 4 day exposure to oncostatin M andAAT production decreasing cytokine effects on the islets (bottom).

FIG. 8A-8D illustrates the effect of AAT on human islets and theproduction of nitric oxide (8A), TNF-α production (8B) IL-6 (8C) andIL-8 (8D).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Definitions

Terms that are not otherwise defined herein are used in accordance withtheir plain and ordinary meaning.

As used herein, “a” or “an” may mean one or more than one of an item.

As used herein “analog of alpha-1-antitrypsin” may mean a compoundhaving alpha-1-antitrypsin-ike activity. In one embodiment, an analog ofalpha-1-antitrypsin is a functional derivative of alpha-1-antitrypsin.In a particular embodiment, an analog of alpha-1-antitrypsin is acompound capable of significantly reducing serine protease activity. Forexample, an inhibitor of serine protease activity has the capability ofinhibiting the proteolytic activity of trypsin, elastase, kallikrein,thrombin, cathepsin G, chymotrypsin, plasminogen activators, plasminand/or other serine proteases.

As used herein “immunomodulatory drugs or agents”, it is meant, e.g.,agents which act on the immune system, directly or indirectly, e.g., bystimulating or suppressing a cellular activity of a cell in the immunesystem, e.g., T-cells, B-cells, macrophages, or antigen presenting cells(APC, dendritic cells), or by acting upon components outside the immunesystem which, in turn, stimulate, suppress, or modulate the immunesystem, e.g. cytokines, e.g., hormones, receptor agonists orantagonists, and neurotransmitters; immunomodulators can be, e.g.,immunosuppressants or immunostimulants.

It is to be understood that the terminology and phraseology employedherein are for the purpose of description and should not be regarded aslimiting

DETAILED DESCRIPTION OF THE INVENTION

In the following sections, various exemplary compositions and methodsare described in order to detail various embodiments of the invention.It will be obvious to one skilled in the art that practicing the variousembodiments does not require the employment of all or even some of thespecific details outlined herein, but rather that concentrations, timesand other specific details may be modified through routineexperimentation. In some cases, well known methods or components havenot been included in the description.

Embodiments of the present invention provide for methods for treating asubject having or in need of a transplant. In accordance with theseembodiments, a subject may be treated with a composition capable ofsignificantly reducing serine protease activity. In addition, oneembodiment of the present invention provides for methods includingtreating a subject with a composition comprising a compound havingα-1-antitrypsin activity. In one embodiment, the composition can includeα-1-antitrypsin, analog thereof or a serine protease inhibitor to forexample, promote transplant survival or reduce a side effect of thetransplant. Further, the administration of the composition can be beforetransplantation, during transplantation, after transplantation orcombination thereof. In addition, the composition may further includeone or more additional therapies such as immunosuppressive therapies. Atransplant of the present invention may include transplantation of anorgan such as lung, kidney, heart, liver, skin, pancreas, or bowel organor non-organ such bone marrow, pancreatic islet, cornea, and/or softtissue.

Serine protease inhibitors, have been found in a variety of organisms.At least nine separate, well-characterized proteins are now identified,which share the ability to inhibit the activity of various proteases.Several of the inhibitors have been grouped together, such as theα₁-antitrypsin-proteinase inhibitor. Serine proteases include but arenot limited to leukocyte elastase, thrombin, cathepsin G, chymotrypsin,plasminogen activators, and plasmin.

Embodiments of the present invention provide for methods for promotingtransplantation, graft survival, reducing graft rejection and/orreducing or preventing side-effects associated with graft rejection. Inaccordance with these embodiments, side-effects may include conditionsassociated with graft versus host disease (GVHD), or graft rejection. Inone example, methods disclosed herein may be used to treat a subjectundergoing bone marrow transplantation. In another embodiment, symptomsor signs may include but is not limited to one or more of the following,malaise, fever, dry cough, myalgias, and chest pains, ventilatorycompromise, sweating, nausea, vomiting, fever, abdominal pain, bloodydiarrhea, mucosal ulcerations, reduced renal function (increasedcreatinine, decreased urine output), reduced pulmonary function(increased shortness of breadth, fever, cough, sputum, hypoxemia),reduced cardiac function (shortness of breach, chest pain, fatigue,pulmonary or peripheral edema, valvulopathy), reduced islet function(increased glucose, diabetes mellitus), graft versus host disease(gastrointestinal (GI) ulceration, pulmonary failure, skin ulceration).

Embodiments of the present invention provide for methods for treating asubject in need of an immunotolerance therapy. In accordance with theseembodiments, a subject may be treated with a composition for inducingimmune tolerance. This achieved while reducing the risk of adysfunctional immune responses or a side-effect of a dysfunctionalimmune response in a subject as typically encountered during standardimmune suppression. For example, a dysfunctional immune response may bean effect of graft rejection, pneumonia, sepsis, fungal infection,cancer. In accordance with this method the subject can be administered acomposition including a compound that is capable of significantlyreducing serine protease activity or other activity associated withα1-antitrypsin or α1-antitrypsin analog. In certain embodiments, acomposition capable of significantly reducing serine protease activitycan include α-1-antitrypsin, an analog thereof or a combination thereof.In accordance with these embodiments, one example for immunotolerancetherapy can include inhibiting cytokine production.

Any of the embodiments detailed herein may further include one or more atherapeutically effective amount of anti-microbial drugsanti-inflammatory agent, immunomodulatory agent, or immunosuppressiveagent or combination thereof.

Non-limiting examples of anti-rejection agents/drugs may include forexample cyclosporine, azathioprine, corticosteroids, FK506 (tacrolimus),RS61443, mycophenolate mofetil, rapamycin (sirolimus), mizoribine,15-deoxyspergualin, and/or leflunomide or any combination thereof.

In addition, other combination compositions of methods disclosed in thepresent invention include certain antibody-based therapies. Non-limitingexamples include, polyclonal anti-lymphocyte antibodies, monoclonalantibodies directed at the T-cell antigen receptor complex (OKT3,TIOB9), monoclonal antibodies directed at additional cell surfaceantigens, including interleukin-2 receptor alpha. Antibody-basedtherapies may be used as induction therapy and/or anti-rejection drugsin combination with the compositions and methods of the presentinvention.

Embodiments of the present invention provide for methods treating asubject in need of an immunotolerance therapy. In accordance with theseembodiments, a subject may be treated with a composition capable ofsignificantly reducing serine protease activity. In one embodiment, thecomposition can include α-1-antitrypsin, analog thereof or a serineprotease inhibitor to for example, to reduce or inhibit the productionof cytokines. In accordance with these embodiments, combinationtherapies are contemplated, such as combining α-1-antitrypsincomposition with an anti-inflammatory agent.

In one particular embodiment, the present inventions provide for methodsfor reducing levels and activities of cytokines such as TNFα (tumornecrosis factor alpha). For example, the composition can includealpha-1-antitrypsin or analog thereof or combination thereof alone or incombination with other therapies.

In one embodiment, the reduction, prevention or inhibition of rejectionof transplantation or side effects thereof associated with one or moreof each of the above-recited conditions may be about 10-20%, 30-40%,50-60%, or more reduction or inhibition due to administration of thedisclosed compositions.

In one embodiment of the present invention a composition may includecompounds that engage molecules for the SEC receptor to treat a subjectundergoing a transplantation and/or in need of immunotolerance therapy.In each of the recited methods, an α1-antitrypsin (e.g. mammalianderived) or inhibitor of serine protease activity substance contemplatedfor use within the methods of the present invention can include a seriesof peptides including carboxyterminal amino acid peptides correspondingto AAT. These pentapeptides can be represented by a general formula (I):I-A-B-C-D-E-F-G-H-II (note: in the Sequence Listing F=X), wherein I isCys or absent; A is Ala, Gly; Val or absent; B is Ala, Gly, Val, Ser orabsent; C is Ser, Thr or absent; D is Ser, Thr, Ans, Glu, Arg, Ile, Leuor absent; E is Ser, Thr, Asp or absent; F is Thr, Ser, Asn, Gln, Lys,Trp or absent; G is Tyr or absent; H is Thr, Gly, Met, Met(O), Cys, Thror Gly; and II is Cys, an amide group, substituted amide group, an estergroup or absent, wherein the peptides includes 4 or more consecutiveamino acids and physiologically acceptable salts thereof. Among thisseries of peptides, several are equally acceptable including FVFLM (SEQID NO. 1), FVFAM (SEQ. ID NO. 2), FVALM (SEQ. ID NO. 3), FVFLA (SEQ. IDNO. 4), FLVFI (SEQ. ID NO. 5), FLMII (SEQ. ID NO. 6), FLFVL (SEQ. ID NO.7), FLFVV (SEQ. ID NO. 8), FLFLI (SEQ. ID NO. 9), FLFFI (SEQ. ID NO.10), FLMFI (SEQ. ID NO. 11), FMLLI (SEQ. ID NO. 12), FIIMI (SEQ. ID NO.13), FLFCI (SEQ. ID NO. 14), FLFAV (SEQ. ID) NO. 15), FVYLI (SEQ. ID NO.16), FAFLM (SEQ. ID NO. 17), AVFLM (SEQ. ID NO. 18), and any combinationthereof.

In several embodiments herein, AAT peptides contemplated for use in thecompositions and methods of the present invention are also intended toinclude any and all of those specific AAT peptides of SEQ ID NO. 1depicted supra. Any combination of consecutive amino acids simulatingAAT or AAT-like activity may be used, such as amino acids 2-12, aminoacids 3-14, 4-16, etc.

In each of the above-recited methods, α1-antitrypsin or analogs thereofare contemplated for use in a composition herein. These analogs mayinclude peptides. The peptides may include but are not limited to aminoacid peptides containing MPSSVSWGIL (SEQ. ID NO. 19); LAGLCCLVPV (SEQ.ID NO. 20) SLAEDPQGDA (SEQ. ID NO. 21); AQKTDTSHHD (SEQ. ID NO. 22)QDHPTFNKIT (SEQ. ID NO. 23); PNLAEFAFSL (SEQ. ID NO. 24); YRQLAHQSNS(SEQ. ID NO. 25); TNIFFSPVSI (SEQ. ID NO. 26); ATAFAMLSLG (SEQ. ID NO.27); TKADTHDEIL (SEQ. ID NO. 28); EGLNFNLTEI (SEQ. ID NO. 29);PEAQIHEGFQ (SEQ. ID) NO. 30); ELLRTLNQPD (SEQ. ID NO. 31); SQLQLTTGNG(SEQ. ID NO. 32); LFLSEGLKLV (SEQ. ID NO. 33); DKFLEDVKKL (SEQ. ID NO.34); YHSEAFTVNF (SEQ. ID NO. 35); GDHEEAKKQI (SEQ. ID NO. 36);NDYVEKGTQG (SEQ. ID NO. 37); KIVDLVKELD (SEQ. ID NO. 38); RDTVFALVNY(SEQ. 1D NO. 39); IFFKGKWERP (SEQ. ID NO. 40); FEVKDTEDED (SEQ. ID NO.41); FHVDQVTTVK (SEQ. ID NO. 42); VPMMKRLGMF (SEQ. ID NO. 43);NIQHCKKLSS (SEQ. ID NO. 44); WVLLMKYLGN (SEQ. ID NO. 45); ATAIFFLPDE(SEQ. ID NO. 46); GKLQHLENEL (SEQ. ID NO. 47); THDIITKFLE (SEQ. ED NO.48); NEDRRSASLH (SEQ. ID NO. 49); LPKLSITGTY (SEQ. ID NO. 50);DLKSVLGQLG (SEQ. ID NO. 51); ITKVFSNGAD (SEQ. ID NO. 52); LSGVTEEAPL(SEQ. ID NO. 53); KLSKAVHKAV (SEQ. ID NO. 54); LTIDEKGTEA (SEQ. ID NO.55); AGAMFLEAIP (SEQ. ID NO. 56); MSIPPEVKFN (SEQ. ID NO. 57);KPFVFLMIEQ (SEQ. ID NO. 58); NTKSPLFMGK (SEQ. ID NO. 59); VVNPTQK (SEQ.ID NO. 60), or any combination thereof.

In Accordance with embodiments of the present invention, the peptide canbe protected or derivitized in by any means known in the art forexample, N-terminal acylation, C-terminal amidation, cyclization, etc.In a specific embodiment, the N-terminus of the peptide is acetylated.

Pharmaceutical Compositions

Embodiments herein provide for administration of compositions tosubjects in a biologically compatible form suitable for pharmaceuticaladministration in vivo. By “biologically compatible form suitable foradministration in vivo” is meant a form of the active agent (i.e.pharmaceutical chemical, protein, gene, antibody etc of the embodiments)to be administered in which any toxic effects are outweighed by thetherapeutic effects of the active agent. Administration of atherapeutically active amount of the therapeutic compositions is definedas an amount effective, at dosages and for periods of time necessary toachieve the desired result. For example, a therapeutically active amountof a compound may vary according to factors such as the disease state,age, sex, and weight of the individual, and the ability of antibody toelicit a desired response in the individual. Dosage regima may beadjusted to provide the optimum therapeutic response.

In one embodiment, the compound (i.e. pharmaceutical chemical, protein,peptide etc. of the embodiments) may be administered in a convenientmanner such as subcutaneous, intravenous, by oral administration,inhalation, transdermal application, intravaginal application, topicalapplication, intranasal or rectal administration. Depending on the routeof administration, the active compound may be coated in a material toprotect the compound from the degradation by enzymes, acids and othernatural conditions that may inactivate the compound. In a preferredembodiment, the compound may be orally administered. In anotherpreferred embodiment, the compound may be administered intravenously. Inone particular embodiment, the compound may be administeredintranasally, such as inhalation.

A compound may be administered to a subject in an appropriate carrier ordiluent, co-administered with enzyme inhibitors or in an appropriatecarrier such as liposomes. The term “pharmaceutically acceptablecarrier” as used herein is intended to include diluents such as salineand aqueous buffer solutions. It may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation. The active agent may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use may beadministered by means known in the art. For example, sterile aqueoussolutions (where water soluble) or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion may be used. In all cases, the composition cant be sterileand can be fluid to the extent that easy syringability exists. It mightbe stable under the conditions of manufacture and storage and may bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The pharmaceutically acceptable carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prevention ofmicroorganisms can be achieved by heating, exposing the agent todetergent, irradiation or adding various antibacterial or antifungalagents.

Sterile injectable solutions can be prepared by incorporating activecompound (e.g. a compound that reduces serine protease activity) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization.

Aqueous compositions can include an effective amount of a therapeuticcompound, peptide, epitopic core region, stimulator, inhibitor, and thelike, dissolved or dispersed in a pharmaceutically acceptable carrier oraqueous medium. Compounds and biological materials disclosed herein canbe purified by means known in the art.

Solutions of the active compounds as free-base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. Prolonged absorption of the injectable compositions canbe brought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above.It is contemplated that slow release capsules, timed-releasemicroparticles, and the like can also be employed. These particularaqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration.

The active therapeutic agents may be formulated within a mixture tocomprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1milligrams, or about 0.1 to 1.0 or even about 1 to 10 gram per dose.Single dose or multiple doses can also be administered on an appropriateschedule for a predetermined condition.

In another embodiment, nasal solutions or sprays, aerosols or inhalantsmay be used to deliver the compound of interest. Additional formulationsthat are suitable for other modes of administration includesuppositories and pessaries. A rectal pessary or suppository may also beused. In general, for suppositories, traditional binders and carriersmay include, for example, polyalkylene glycols or triglycerides; suchsuppositories may be formed from mixtures containing the activeingredient in the range of 0.5% to 10%, preferably 1%-2%.

Oral formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate and thelike. In certain defined embodiments, oral pharmaceutical compositionswill comprise an inert diluent or assimilable edible carrier, or theymay be enclosed in hard or soft shell gelatin capsule, or they may becompressed into tablets, or they may be incorporated directly with thefood of the diet. For oral therapeutic administration, the activecompounds may be incorporated with excipients and used in the form ofingestible tablets, buccal tables, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 0.1% of active compound. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 75% of theweight of the unit, or preferably between 25-60%. The amount of activecompounds in such therapeutically useful compositions is such that asuitable dosage will be obtained.

A pharmaceutical composition may be prepared with carriers that protectactive ingredients against rapid elimination from the body, such astime-release formulations or coatings. Such carriers include controlledrelease formulations, such as, but not limited to, microencapsulateddelivery systems, and biodegradable, biocompatible polymers, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid,polyorthoesters, polylactic acid and others are known.

Pharmaceutical compositions are administered in an amount, and with afrequency, that is effective to inhibit or alleviate side effects of atransplant and/or to reduce or prevent rejection. The precise dosage andduration of treatment may be determined empirically using known testingprotocols or by testing the compositions in model systems known in theart and extrapolating therefrom. Dosages may also vary with the severityof the condition. A pharmaceutical composition is generally formulatedand administered to exert a therapeutically useful effect whileminimizing undesirable side effects. In general, an oral dose rangesfrom about 200 mg to about 1000 mg, which may be administered forexample 1 to 3 times per day.

It will be apparent that, for any particular subject, specific dosageregimens may be adjusted over time according to the individual need. Thepreferred doses for administration can be anywhere in a range betweenabout 0.01 mg and about 100 mg per ml of biologic fluid of treatedpatient. In one particular embodiment, the range can be between 1 and100 mg/kg which can be administered daily, every other day, biweekly,weekly, monthly etc. In another particular embodiment, the range can bebetween 10 and 75 mg/kg introduced weekly to a subject. Thetherapeutically effective amount of α1-antitrypsin, peptides, or drugsthat have similar activities as α1-antitrypsin or peptides can be alsomeasured in molar concentrations and can range between about 1 nM toabout 2 mM.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, as gum tragacanth, acacia, cornstarch, or gelatin;excipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; and a sweetening agent, such as sucrose, lactoseor saccharin may be added or a flavoring agent.

Liposomes can be used as a therapeutic delivery system and can beprepared in accordance with known laboratory techniques. In addition,dried lipids or lyophilized liposomes prepared as previously describedmay be reconstituted in a solution of active agent (e.g. nucleic acid,peptide, protein or chemical agent), and the solution diluted to anappropriate concentration with a suitable solvent known to those skilledin the art. The amount of active agent encapsulated can be determined inaccordance with standard methods.

In a preferred embodiment, a nucleic acid (e.g. α1-antitrypsin oranalogs thereof) and the lipid dioleoylphosphatidylcholine may beemployed. For example, nuclease-resistant oligonucleotides may be mixedwith lipids in the presence of excess t-butanol to generateliposomal-oligonucleotides for administration.

The pharmaceutical compositions containing the α1-antitrypsin, analogthereof, or inhibitor of serine protease activity or a functionalderivative thereof may be administered to individuals, particularlyhumans, for example by subcutaneously, intramuscularly, intranasally,orally, topically, transdermally, parenterally, gastrointestinally,transbronchially and transalveolarly. Topical administration isaccomplished via a topically applied cream, gel, rinse, etc. containingtherapeutically effective amounts of inhibitors of serine proteases.Transdermal administration is accomplished by application of a cream,rinse, gel, etc. capable of allowing the inhibitors of serine proteasesto penetrate the skin and enter the blood stream. In addition, osmoticpumps may be used for administration. The necessary dosage will varywith the particular condition being treated, method of administrationand rate of clearance of the molecule from the body.

In each of the aforementioned compositions and methods, a compoundhaving serine protease inhibitor activity and/or having α1-antitrypsinactivity or analog thereof may be used in a single therapeutic dose,acute manner or a chronic manner to treat episodes or prolonged bouts,respectively, in promoting graft survival, treating graft rejectionand/or associated graft rejection-induced side-effects.

In certain embodiments of the methods of the present invention, thesubject may be a mammal such as a human or a veterinary and/or adomesticated animal.

Therapeutic Methods

In one embodiment of the present invention, methods provide for treatinga subject in need of or undergoing a transplant. For example, treatmentsfor reducing graft rejection, promoting graft survival, and promotingprolonged graft function by administering to a subject in need thereof atherapeutically effective amount of a composition. The composition caninclude a compound capable of inhibiting at least one serine proteasefor example, al-antitrypsin, or analog thereof.

Preserving the Craft During Transplant Before Engraftment

According to the methods of the present invention, transplantationcomplications can be reduced or inhibited to obtain importanttherapeutic benefits. Therefore, administration of a therapeuticcomposition contemplated by embodiments of the invention, i.e.,α1-antitrypsin, derivative or analog thereof, can be beneficial for thetreatment of transplantation complications or conditions.

Another beneficial effect of use of the compositions and methods of thepresent invention include reducing negative effects on an organ ornon-organ during explant, isolation, transport and/or prior toimplantation. For example, the composition can reduce apoptosis, reduceproduction of cytokines, reduce production of NO, or combination thereofin an organ for transplant. In one particular embodiment, a compositioncan include a compound that includes alpha-1-antitrypsin, an analogthereof, a serine protease inhibitor, serine protease inhibitor-likeactivity, analog thereof or a combination thereof. The transplant organor non-organ can include but is not limited to, lung, kidney, heart,liver, soft tissue, skin, pancreas, intestine, soft tissue cornea, bonemarrow, stem cell, pancreatic islet, and combination thereof.

In a further embodiment, the methods and compositions of the inventionare useful in the therapeutic treatment of graft rejection associatedside effects. In a yet further embodiment, graft rejection associatedside effects can be prevented by the timely administration of the agentof the invention as a prophylactic, prior to onset of one or moresymptoms, or one or more signs, or prior to onset of one or more severesymptoms or one or more signs of a graft rejection associated disease.Thus, a patient at risk for a particular graft rejection or graftrejection-associated disease or clinical indication can be treated withserine protease inhibitors, for example,(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(3-Trifluoromethylbenzyl)-1,2,4-oxadiazolyl)carbonyl)-2-(S)-Methylpropyl]-L-Prolinamide;as a prophylactic measure.

It is contemplated herein that the present compositions and methods ofthe present invention can be used to treat patients with one or moregrafts who require chronic therapy to maintain graft integrity, and suchpatients will therefore benefit from indefinite or chronic use of therejection repressive therapy of the methods of the present invention.Yet another embodiment can be used to treat flairs of acute rejection soas to minimize the effects of acute clinical rejection, organ failure,and/or eventual destruction of the graft.

Desirable blood levels may be maintained by continuous infusion toprovide about 0.01-5.0 mg/kg/hr or by intermittent infusions containingabout 0.4-20 mg/kg of the active ingredient(s). Buffers, preservatives,antioxidants and the like can be incorporated as required. It isintended herein that the ranges recited also include all those specificpercentage amounts between the recited range. For example, the range ofabout 0.4 to 20 mg/kg also encompasses 0.5 to 19.9%, 0.6 to 19.8%, etc,without actually reciting each specific range therewith,

Serine Protease Inhibitors

It is to be understood that the present invention is not limited to theexamples described herein, and other serine proteases known in the artcan be used within the limitations of the invention. For example, oneskilled in the art can easily adopt inhibitors as described in WO98/24806, which discloses substituted oxadiazole, thiadiazole andtriazole as serine protease inhibitors. U.S. Pat. No. 5,874,585discloses substituted heterocyclic compounds useful as inhibitors ofserine proteases for example,(benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(3-trifluoromethylbenzyl)-1,2,4-oxadiazolyl)carbonyl)-2-(S)-methylpropyl]-L-prolinamidebenzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(2-phenylethyl)-1,2,4-oxadiazolyl)carbonyl)-2-(S)-methylpropyl]-L-prolinamide;and(benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(2-methoxybenzyl)-1,2,4-oxadiazolyl)carbonyl)-2-(S)-methylpropyl]-L-prolinamide.

α1-antitrypsin is a glycoprotein of MW 51,000 with 417 amino acids and 3oligosaccharide side chains. Human α1-antitrypsin is a singlepolypeptide chain with no internal disulfide bonds and only a singlecysteine residue normally intermolecularly disulfide-linked to eithercysteine or glutathione. The reactive site of α1-antitrypsin contains amethionine residue, which is labile to oxidation upon exposure totobacco smoke or other oxidizing pollutants. Such oxidation reduces theelastase-inhibiting activity of α1-antitrypsin; therefore substitutionof another amino acid at that position, i.e. alanine, valine, glycine,phenylalanine, arginine or lysine, produces a form of α1-antitrypsinwhich is more stable. α1-antitrypsin can be represented by the followingformula: SEQ ID 61:

MPSSVSWGIL LAGLCCLVPV SLAEDPQGDA AQKTDTSHHD QDHPTFNKITPNLAEFAFSL 100YRQLAHQSNS TNIFFSPVSI ATAFAMLSLG TKADTHDEILEGLNFNLTEI PEAQIHEGFQ ELLRTLNQPD SQLQLTTGNG LFLSEGLKLVDKFLEDVKKL 200YHSEAFTVNF GDHEEAKKQI NDYVEKGTQG KIVDLVKELDRDTVFALVNY IFFKGKWERP FEVKDTEDED FHVDQVTTVK VPMMKRLGMF NIQHCKKLSS 300WVLLMKYLGN ATAIFFLPDE GKLQHLENEL THDIITKFLENEDRRSASLH LPKLSITGTY DLKSVLGQLG ITKVFSNGAD LSGVTEEAPL KLSKAVHKAV 400LTIDEKGTEA AGAMFLEAIP MSIPPEVKFN KP FVFLM IEQ NTKSPLFMGK VVNPTQK 417

One important amino acid sequence near the carboxyterminal end ofα1-antitrypsin is shown in bold and underlined and is pertinent to thisinvention (details of the sequence can be found for example in U.S. Pat.No. 5,470,970, as incorporated by reference).

Extrahepatic sites of AAT production include neutrophils, monocytes andmacrophages, and the expression of AAT is inducible in response to LPS,TNFα, IL-1 and IL-6 in various cell types. Deficiency in AAT isassociated with immune dysfunctional conditions such as rheumatoidarthritis and systemic lupus erythematosus.

Other serine protease inhibitor molecules, which may be used in any ofthe disclosed compositions may include compounds disclosed in thefollowing: WO 98/20034 disclosing serine protease inhibitors from fleas;WO98/23565 disclosing aminoguanidine and alkoxyguanidine compoundsuseful for inhibiting serine proteases; WO98/50342 disclosingbis-aminomethylcarbonyl compounds useful for treating cysteine andserine protease disorders; WO98/50420 cyclic and other amino acidderivatives useful for thrombin-related diseases; WO 97/21690 disclosingD-amino acid containing derivatives; WO 97/10231 disclosingketomethylene group-containing inhibitors of serine and cysteineproteases; WO 97/03679 disclosing phosphorous containing inhibitors ofserine and cysteine proteases; WO 98/21186 benzothiazo and relatedheterocyclic inhibitors of serine proteases; WO 98/22619 disclosing acombination of inhibitors binding to P site of serine proteases withchelating site of divalent cations; WO 98/22098 disclosing a compositionwhich inhibits conversion of pro-enzyme CPP32 subfamily includingcaspase 3 (CPP32/Yama/Apopain); WO 97/48706 disclosingpyrrolo-pyrazine-diones; and WO 97/33996 disclosing human placentalbikunin (recombinant) as serine protease inhibitor.

Other compounds having serine protease inhibitory activity are equallysuitable and effective for use in the methods of the present invention,including but not limited to: tetrazole derivatives as disclosed in WO97/24339; guanidinobenzoic acid derivatives as disclosed in WO 97/37969and in a number of U.S. Pat. Nos. 4,283,418; 4,843,094; 4,310,533;4,283,418; 4,224,342; 4,021,472; 5,376,655; 5,247,084; and 5,077,428;phenylsulfonylamide derivatives represented by general formula in WO97/45402; novel sulfide, sulfoxide and sulfone derivatives representedby general formula in WO 97/49679; novel amidino derivatives representedby general formula in WO 99/41231; other amidinophenol derivatives asdisclosed in U.S. Pat. Nos. 5,432,178; 5,622,984; 5,614,555; 5,514,713;5,110,602; 5,004,612; and 4,889,723 among many others.

Graft Rejection and Graft Survival-Side-Effects and Conditions

One of the beneficial effects of use of the compositions and methods ofthe present invention include, for example, and not by way oflimitation, reduced infiltration of graft with cells or serum factors(including but not limited to, complement, anti graft antibody thatgenerate inflammation and graft rejection), reduced cytokines, reducednitric oxide, reduced apoptosis, and reduced specific immune responseagainst the graft or any combination thereof.

Management of Graft Rejection

By preventing or reducing the side effects or conditions associated withgraft survival or graft rejection using this novel approach, severaladvantages are obtained compared to alternative approaches, for example,and not by way of limitation:

1. Reduced infiltration of graft with cells or serum factors (forexample, and not by way of limitation, complement, anti graft antibodythat generate inflammation and graft rejection); reduced production ofcytokines or nitric oxide (NO) that can induce inflammation orapoptosis; inhibits apoptosis; inhibits immune activation, inhibits CMVor any combination thereof.

2. Synthetic inhibitors of serine proteases (AAT-like mimics or analogs)can and have been developed by means known in the art. Such apharmaceutical agent may be formulated as for example, a cream to treatgraft rejection and/or promote graft survival.

3. Commercially available agents already approved for different use inhumans will work as a treatment for graft rejection and/or promote graftsurvival. These agents are currently used for indications other thangraft rejection and/or to promote graft survival, and include injectibleAAT, plasma preparations, aprotinin and others (American J. of RespCritical Care Med 1998, V11 158: 49-59, incorporated herein by referencein its entirety). In one embodiment, serine protease inhibitors may bedelivered by inhalation. An inhaled agent (natural AAT or a syntheticAAT-like mimic/or other inhibitor of serine protease) may be especiallyuseful due to elevated local concentrations, ease of drug delivery, andlack of side effects (since administration is not systemic). This modeof focused drug delivery may augment serine protease inhibitor activitywithin the lung tissues and associated lymphatics, which are two of theprincipal sites where diseases and/or clinical conditions associatedwith graft rejection and/or promotion of graft survival develop.

4. By promoting graft survival and/or treating graft rejection, thedirect cause of the side effect is disrupted in affected individuals.This invention specifically contemplates inhibiting host cell serineproteases or induce the SEC receptor or combination thereof as a methodof treating graft rejection and/or promoting graft survival in a mammalin need thereof in conjunction with administration of one or moreanti-rejection and/or anti-microbial.

5. There is an extensive clinical experience using injectible AAT totreat patients with genetic AAT deficiency. No long-term negativeeffects have been detected to date (American J. of Resp Critical CareMed 1998, V11 158: 49-59; Wencker et al. Chest 2001 119:737-744).Moreover, a small molecule inhibitor of host serine protease has beenadministered to patients with Kawasaki's Disease (Ulinistatin, Onopharmaceuticals).

Isolated Proteins for Use in the Compositions and Methods of theInvention

One aspect of the invention pertains to proteins, and portions thereof,as well as polypeptide fragments suitable for use as immunogens to raiseantibodies directed against a polypeptide of the invention. In oneembodiment, the native polypeptide can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, polypeptides of theinvention are produced by recombinant DNA techniques. Alternative torecombinant expression, a polypeptide of the invention can besynthesized chemically using standard peptide synthesis techniques.

Recombinant unmodified and mutant variants of .alpha.sub.1-antitrypsinproduced by genetic engineering methods are also known (see U.S. Pat.No. 4,711,848). The nucleotide sequence of human alpha.sub.1-antitrypsinand other human alpha.sub.1-antitrypsin variants has been disclosed ininternational published application No. WO 86/00,337, the entirecontents of which are incorporated herein by reference. This nucleotidesequence may be used as starting material to generate all of the AATamino acid variants and amino acid fragments depicted herein, usingrecombinant DNA techniques and methods known to those of skill in theart.

An isolated and/or purified or partially purified protein orbiologically active portion thereof may be used in any embodiment of theinvention. A protein that is substantially free of cellular materialincludes preparations of protein having less than about 30%, 20%, 10%,or 5% (by dry weight) of heterologous protein. When the protein orbiologically active portion thereof is recombinantly produced, it canalso be substantially free of culture medium. When the protein isproduced by chemical synthesis, it is preferably substantially free ofchemical precursors or other chemicals. Accordingly, such preparationsof the protein have less than about 30%, 20%, 10%, and 5% (by dryweight) of chemical precursors or compounds other than the polypeptideof interest.

Biologically active portions of a polypeptide of the invention includepolypeptides including amino acid sequences sufficiently identical to orderived from the amino acid sequence of the protein (e.g., the aminoacid sequence shown in any of SEQ ID Nos: 1 to 60, which exhibit atleast one activity of the corresponding full-length protein). Abiologically active portion of a protein of the invention can be apolypeptide, which is, for example, 5, 10, 25, 50, 100 or more aminoacids in length. Moreover, other biologically active portions, in whichother regions of the protein are deleted, can be prepared by recombinanttechniques and evaluated for one or more of the functional activities ofthe native form of a polypeptide of the invention.

Preferred polypeptides have the amino acid sequence of SEQ ID Nos: 1 to60. Other useful proteins are substantially identical (e.g., at leastabout 45%, preferably 55%, 65%, 75%, 85%, 95%, or 99%) to any of SEQ IDNOs: 1 to 60, and retain the functional activity of the protein of thecorresponding naturally-occurring protein yet differ in amino acidsequence due to natural allelic variation or mutagenesis.

The compounds of the present invention can be used as therapeutic agentsin the treatment of a physiological (especially pathological) conditioncaused in whole or part, by excessive serine protease activity. Inaddition, a physiological (especially pathological) condition can beinhibited in whole or part. Peptides contemplated herein may beadministered as free peptides or pharmaceutically acceptable saltsthereof. The peptides should be administered to individuals as apharmaceutical composition, which, in most cases, will include thepeptide and/or pharmaceutical salts thereof with a pharmaceuticallyacceptable carrier.

When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the defaultparameters of the respective programs (e.g., XBLAST and NBLAST) can beused. See http://www.ncbi.nlm.nih.gov.

The present invention also pertains to variants of the polypeptides ofthe invention. Such variants have an altered amino acid sequence whichcan function as either agonists (mimetics) or as antagonists. Variantscan be generated by mutagenesis, e.g., discrete point mutation ortruncation. An agonist can retain substantially the same, or a subset,of the biological activities of the naturally occurring form of theprotein. An antagonist of a protein can inhibit one or more of theactivities of the naturally occurring form of the protein by, forexample, competitively binding to a downstream or upstream member of acellular signaling cascade which includes the protein of interest. Thus,specific biological effects can be elicited by treatment with a variantof limited function. Treatment of a subject with a variant having asubset of the biological activities of the naturally occurring form ofthe protein can have fewer side effects in a subject relative totreatment with the naturally occurring form of the protein.

Variants of a protein of the invention which function as either agonists(mimetics) or as antagonists can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of theprotein of the invention for agonist or antagonist activity.

Fusion Polypeptides

In other embodiments, compounds having serine protease inhibitoractivity such as α1-antitrypsin and/or analog thereof, may be part of afusion polypeptide. In one example, a fusion polypeptide may includeα1-antitrypsin (e.g. mammalian α1-antitrypsin) or an analog thereof anda different amino acid sequence that may be heterologous to theα1-antitrypsin or analog substance.

In yet other embodiments, the fusion polypeptide contemplated for use inthe methods of the present invention can additionally include an aminoacid sequence that is useful for identifying, tracking or purifying thefusion polypeptide, e.g., a FLAG or HIS tag sequence. The fusionpolypeptide can include a proteolytic cleavage site that can remove theheterologous amino acid sequence from the compound capable of serineprotease inhibition, such as mammalian α1-antitrypsin or analog thereof.

In one embodiment, fusion polypeptides of the invention are produced byrecombinant DNA techniques. Alternative to recombinant expression, afusion polypeptide of the invention can be synthesized chemically usingstandard peptide synthesis techniques. The present invention alsoprovides compositions that comprise a fusion polypeptide of theinvention and a pharmaceutically acceptable carrier, excipient ordiluent.

In particular, in one embodiment the fusion protein comprises aheterologous sequence that is a sequence derived from a member of theimmunoglobulin protein family, for example, comprise an immunoglobulinconstant region, e.g., a human immunoglobulin constant region such as ahuman IgG1 constant region. The fusion protein can, for example, includea portion of α1-antitrypsin, analog thereof or inhibitor of serineprotease activity polypeptide fused with the amino-terminus or thecarboxyl-terminus of an immunoglobulin constant region, as disclosed,e.g., in U.S. Pat. No. 5,714,147, and U.S. Pat. No. 5,116,964. Inaccordance with these embodiments, the FcR region of the immunoglobulinmay be either wild-type or mutated. In certain embodiments, it isdesirable to utilize an immunoglobulin fusion protein that does notinteract with an Fc receptor and does not initiate ADCC reactions. Insuch instances, the immunoglobulin heterologous sequence of the fusionprotein can be mutated to inhibit such reactions. See, e.g., U.S. Pat.No. 5,985,279 and WO 98/06248.

In yet another embodiment, α1-antitrypsin, analog thereof, or inhibitorof serine protease activity polypeptide fusion protein comprises a GSTfusion protein in which is fused to the C-terminus of GST sequences.Fusion expression vectors and purification and detection means are knownin the art.

Expression vectors can routinely be designed for expression of a fusionpolypeptide of the invention in prokaryotic (e.g., E. coli) oreukaryotic cells (e.g., insect cells (using baculovirus expressionvectors), yeast cells or mammalian cells) by means known in the art.

Expression of proteins in prokaryotes may be carried out by means knownin the art. Such fusion vectors typically serve three purposes: 1) toincrease expression of recombinant protein; 2) to increase thesolubility of the recombinant protein; and 3) to aid in the purificationof the recombinant protein by acting as a ligand in affinitypurification.

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector as described inthe art. In another embodiment, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid) such aspancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166). Ahost cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell(e.g., insect cells, yeast or mammalian cells). Vector DNA can beintroduced into prokaryotic or eukaryotic cells via conventionaltransformation or transfection techniques.

Combination Therapies

In each of the aforementioned methods of the present invention, the useof a compound capable of inhibiting serine protease or α1-antitrypsin oranalog thereof alone or in combination with standard immunosuppressiveagents enables transplantation of grafts into immunosuppressed orimmunocompromised recipients. This combination therapy will expand theeligible patient population able to receive this form of treatment.

In each of the aforementioned aspects and embodiments of the invention,combination therapies other than those already enumerated above are alsospecifically contemplated herein. In particular, the compositions of thepresent invention may be administered with one or more macrolide ornon-macrolide antibiotics, anti-bacterial agents, anti-fungals,anti-viral agents, and anti-parasitic agents. Examples of macrolideantibiotics that may be used in combination with the composition of thepresent invention include but are not limited to synthetic,semi-synthetic or naturally occurring macrolidic antibiotic compounds:methymycin, neomethymycin, YC-17, litorin, TMP-SSX, erythromycin A to F,and oleandomycin. Examples of preferred erythromycin anderythromycin-like compounds include: erythromycin, clarithromycin,azithromycin, and troleandomycin.

Examples of anti-bacterial agents include, but are not limited to,penicillins, quinolonses, aminoglycosides, vancomycin, monobactams,cephalosporins, carbacephems, cephamycins, carbapenems, and monobactamsand their various salts, acids, bases, and other derivatives.

Anti-fungal agents include, but are not limited to, caspofungin,terbinafine hydrochloride, nystatin, and selenium sulfide.

Anti-viral agents include, but are not limited to, gancyclovir,acyclovir, valacylocir, amantadine hydrochloride, rimantadin andedoxudine

Examples of macrolide antibiotics that may be used in combination withthe composition of the present invention include but are not limited tosynthetic, semi-synthetic or naturally occurring macrolidic antibioticcompounds: methymycin, neomethymycin, YC-17, litorin, TMP-SSX,erythromycin A to F, and oleandomycin. Examples of preferrederythromycin and erythromycin-like compounds include: erythromycin,clarithromycin, azithromycin, and troleandomycin.

Anti-parasitic agents include, but are not limited to,pirethrins/piperonyl butoxide, permethrin, iodoquinol, metronidazole,co-trimoxazole (sulfamethoxazole/trimethoprim), and pentamidineisethionate.

In another aspect, in the method of the present invention, one may, forexample, supplement the composition by administration of atherapeutically effective amount of one or more an anti-inflammatory orimmunomodulatory drugs or agents. By “anti-inflammatory drugs”, it ismeant, e.g., agents which treat inflammatory responses, i.e., a tissuereaction to injury, e.g., agents which treat the immune, vascular, orlymphatic systems.

Anti-inflammatory or immunomodulatory drugs or agents suitable for usein this invention include, but are not limited to, interferonderivatives, (e.g., betaseron); prostane derivatives, (e.g., compoundsdisclosed in PCT/DE93/0013, iloprost, cortisol, dexamethasone;immunsuppressives, (e.g., cyclosporine A, FK-506 (mycophenylatemofetil); lipoxygenase inhibitors, (e.g., zileutone, MK-886, WY-50295);leukotriene antagonists, (e.g., compounds disclosed in DE 40091171German patent application P 42 42 390.2); and analogs; peptidederivatives, (e.g., ACTH and analogs); soluble TNF-receptors;TNF-antibodies; soluble receptors of interleukins, other cytokines,T-cell-proteins; antibodies against receptors of interleukins, othercytokines, and T-cell-proteins.

Kits

In still further embodiments, the present invention concerns kits foruse with the methods described above. Small molecules, proteins orpeptides may be employed for use in any of the disclosed methods. Inaddition, other agents such as anti-bacterial agents, immunosuppressiveagents, anti-inflammatory agents may be provided in the kit. The kitswill thus can include, in suitable container means, a protein or apeptide or analog agent, and optionally one or more additional agents.

The kits may further include a suitably aliquoted composition of theencoded protein or polypeptide antigen, whether labeled or unlabeled, asmay be used to prepare a standard curve for a detection assay.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, syringe or other container means, intowhich the antibody or antigen may be placed, and preferably, suitablyaliquoted. Where a second or third binding ligand or additionalcomponent is provided, the kit will also generally contain a second,third or other additional container into which this ligand or componentmay be placed. The kits of the present invention will also typicallyinclude a means for containing the antibody, antigen, and any otherreagent containers in close confinement for commercial sale. Suchcontainers may include injection or blow-molded plastic containers intowhich the desired vials are retained.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1

Alpha-1-Antitrypsin Prolongs Graft Islet Graft Survival in Mice

FIG. 1A-1D. Islets from DBA/2 mice (H-2d) were transplanted under therenal capsule of streptozotocin-induced hyperglycemic C57BL/6 mice(H-2b). (A) Glucose levels from days 6-18. Control consists of mice thatwere untreated (n=3) or treated from day −1 every 3 days with humanalbumin (ALB, 6 mg, n=3). Prolonged islet graft survival is observed inmice treated from day −1 every 3 days with human AAT (2 mg, n=10).*P<0.05, **P<0.01, ***P<0.001 between glucose levels on same day. (B)Treatment protocols. Control and full AAT treatment are described inpanel A. Early AAT treatment consists of treatment on days −1, 1 and 3(2 mg, n=3). Late AAT treatment consists of treatment from day 2 and onevery 2 days (2 mg, n=3). Rejection indicates the day that glucoselevels exceed 300 mg/dl. (C) Effect of mouse anti-human-AAT antibodies.Dashed line indicates post transplantation glucose levels of a mouseunder full AAT treatment protocol (see A, B) that was immunized bymultiple administrations of human AAT prior to transplantation (1representative, n=3). Solid line indicates glucose levels of anon-immunized mouse treated under full AAT treatment protocol (1representative, n=10). Arrow indicates detection of treatment-induced,anti-human-AAT antibodies in the non-immunized representative mouse. (D)Comparison of day 15 post-transplantation glucose levels in mice thatwere under full treatment protocol with ALB (n=3) or AAT (non-immunizedn=10, immunized n=3). Of the AAT-treated group, antibodies were detectedon day 15 in 3/3 immunized mice and in 6/10 non-immunized mice.**P=0.005 between mice that produced antibodies (n=6) and mice that didnot produce antibodies (n=4).

Treatment with human albumin (6 mg) resulted in graft rejectioncomparable to that of untreated recipient mice. In contrast, recipientmice that received AAT (2 mg) exhibited prolonged graft function. Asdepicted in FIG. 1 b, neither of the partial treatment protocols, i.e.,days −1, 1 and 3 (‘early treatment’) or days 2 and beyond (‘latetreatment’) prolonged allograft survival.

AAT-treated mice developed anti-human-AAT antibodies (FIGS. 1C and D).Individual mice exhibited anti-human-AAT antibodies at various timepoints (data not shown). To ascertain that the antibodies reduce theprotective effect of AAT, a group of mice was pre-exposed (“immunized”)to human AAT two months before being rendered hyperglycemic andtransplanted with allogeneic islets. These graft recipients were treatedwith the full AAT protocol, despite exhibiting high titers of specificantibodies before engraftment, and displayed rapid graft rejection (FIG.1C). Day 15 was chosen to depict an association between antibodyformation and loss of AAT protective activity; at this time pointAAT-treated mice were divided into positive and negative producers ofanti-human-AAT antibodies. As shown in FIG. 1D, on day 15 allantibody-positive mice were hyperglycemic and all antibody-negative micewere normoglycemic.

Example 2

FIG. 2A-2D illustrates an exemplary method of the effect of AAT onthioglycolate-elicited peritoneal cellular infiltrates. Mice wereadministered intraperitoneal 0.1 ml saline, ALB, AAT or oxidized-AATfollowed by 1 ml of saline or thioglycolate (ThG, 3% w/v, n=3 pergroup). Peritoneal lavage was performed on separate groups after 24 and48 hours. (A) Total cell population of lavaged cells of (open bars)saline or (closed bars) AAT-treated (5 mg) thioglycolate-injected mice.**P<0.05. (B) Percent cell population from saline-treated mice at 48hours. **P<0.05. (C) Oxidation of AAT. AAT was subjected to oxidativeradicals (see Methods). Loss of serine protease activity of oxidized AATwas assessed in an elastase assay. Activity of elastase in the absenceof native AAT was set at 100% and the percentage of activity in thepresence of native and oxidized AAT was calculated (n=3). ***P<0.001. InFIG. 2D, elicited macrophages and neutrophils are identified. Peritonealinfiltrates from 48 hour lavages of ALB (6 mg) and AAT-treated (6 mg),thioglycolate-injected mice were stained for FACS analysis by specificantibodies. Macrophages and neutrophils were identified on the basis ofF4/80 and GR1 versus side scatter flow cytometry profiles. Top, FACSanalysis representative graphs (n=3). Quantified FACS results (n=3) aredepicted in the bottom.

AAT Inhibits Cellular Infiltration

To address the possibility that AAT affects effector cell infiltration,two models of cell emigration were examined: thioglycolate(ThG)-elicited peritoneal infiltration, and cellular infiltration due tointraperitoneal injection of MHC-incompatible fibroblasts.

As shown in FIG. 2A, there was a progressive increase in total cellcount at 24 and 48 hours in mice injected with ThG, whereas nosignificant increase was observed in mice injected with AAT and ThG. At48 hours, total cell count in peritoneal lavage of AAT-treated mice was50% of that of control (FIG. 2B). Total cell count in mice that receivedalbumin control was similar to that of saline-treated mice. There was adose-dependent effect of AAT in that one-sixth the dose was found toreduce cell count to a lesser extent in a significant manner. OxidizedAAT, which had lost its in vitro anti-elastase activity (FIG. 2C), didnot affect cellular infiltrate at 1 mg (FIG. 2B).

The decrease in total cell count is primarily attributed to a decreasein the number of neutrophils (FIG. 2D), identified by theirGR-1high/intermediate side-scatter (SSC) profile. No major differencewas observed with the infiltration of macrophages, identified by theirF4/80int, GR-1int, intermediate SSC profile¹², which is distinct fromthe F4/80very high, GR-1low, high SSC profile of resident macrophages¹²(data not shown).

Example 3

FIG. 3A-3C illustrates an exemplary method of the effect of AAT onMHC-incompatible, NIH-3T3-fibroblast-elicited peritoneal cellularinfiltrates. Mice (C57BL/6; H-2b) were injected i.p. 0.1 ml saline orAAT (1 mg) followed by 1 ml NIH-3T3 cells (1′107 cells in saline; H-2d).Peritoneal lavage was performed daily on days 1-5 and cellsubpopulations were identified by FACS analysis. (n=3 per treatment).(A) Cell numbers. The number of cells in each subpopulation wascalculated from the percentages obtained by FACS analysis, and totalnumber of cells in the infiltrate. *P<0.05, **P<0.01 between cellnumbers on the same day. (B) Representative FACS analysis. (C) Effect ofAAT on intensity and function of infiltrate elicited by islet allograft.Left, Hematoxilyn and Eosin (H&E) staining of day 7 islet allografts. Asection of AAT-treated islet graft (white frame) is compared to asimilar section of ALB-treated diabetic recipient mouse (full treatmentprotocol, see FIG. 1A). Arrow points at border between islet andsurrounding infiltrate. Right, Immunohistochemistry (IHC) withanti-insulin antibodies of day 15 islet grafts. A section of autologousislet graft (white frame) is compared to similar sections of allograftsof AAT- and ALB-treated recipient mice. R, renal parenchyma, G, graft,C, renal capsule.

As illustrated in FIG. 3A, introduction of allogeneic cells evoked acellular infiltrate that consisted of early appearing neutrophils andactivated macrophages, and late appearing CD3+ and NK cells (FIG. 3B).AAT-treated mice exhibited a reduction in neutrophils, CD3+ and NKcells, dark color is insulin staining

To evaluate the level of cellular infiltration into grafted islets,grafts from AAT- and ALB-treated recipient mice were removed on day 7,fixed in paraformaldehyde and stained with Hematoxilin and Eosin. Asdepicted in FIG. 3C (left), a cellular infiltrate is demonstrableregardless of AAT treatment, and includes neutrophils and lymphocytes.However, the infiltrates evoked by grafts of ALB-treated recipient micewere more massive and cause the disruption of islet borders, compared tointact islets of AAT-treated recipient mice. To evaluate islet function,grafts from AAT- and ALB-treated recipient mice were removed on day 15,and immunohistochemistry was performed with anti-insulin antibodies,dark color is insulin staining As depicted in FIG. 3C (right), insulinproduction is preserved on day 15 in islets of AAT-treated recipients.

Example 4

FIG. 4A-4H illustrates an exemplary method of the effect of AAT on isletresponses. (A-D) Islets from C57BL/6 mice were cultured at 100islets/well, in duplicate. AAT was incubated at the indicatedconcentrations for 1 hour before the addition of IFNγ (5 ng/ml) plusIL-1β (10 ng/ml). 72 hours later, supernatants were collected and isletviability was assessed. Islet cells responses in the absence of AAT wereset at 100%. Data are combined from 3 individual experiments, induplicate. **P<0.01, ***P<0.001 between AAT-treated and untreatedislets. Mean±SEM of a. nitrite levels, b. Cell viability and c. MIP-1αlevels. Dashed line represents islets incubated at one-30th theconcentration of IFNγ/IL-1β. d. TNFα levels. (E) Insulin inductionassay. Islets were incubated in triplicate (20 islets/well) in thepresence of AAT (0.5 mg/ml) or ALB (0.5 mg/ml) 1 hour before addition ofIFNγ (5 ng/ml) plus IL-1β (10 ng/ml). 24 hours later, islets weretransferred to a 3 mM or 20 mM glucose solution for 30 minutes andinsulin levels were measured. Vertical axis depicts the ratio betweeninsulin levels at both glucose concentrations. *P<0.05 betweenAAT-treated and ALB-treated islets. (F) Streptozotocin toxicity. C57BL/6mice were injected i.p. with AAT (5 mg) or saline, one day before, onsame day and one day after injection of streptozotocin (225 mg/kg) orsaline (n=3 per group). 48 hours later, pancreata were removed andinsulin-containing cells were identified by immunohistochemistry. Eachimage depicts a representative islet form one pancreas. Graph, mean±SEMpercent change of insulin-containing cells as determined manually fromimages of 2 islets per pancreas (n=6 per treatment group). *P<0.05. (G)Cellular content of islets. Freshly isolated islets (100 islets intriplicate) and residual non-islet pancreatic debris were dissociatedinto single cell suspensions and stained for FACS analysis withanti-CD45-APC or isotype control antibody. Shaded area, islets. Openarea, debris. (H) MHC class II expression. Islets from C57BL/6 mice werecultured (100 islets/well in duplicate) in the presence of AAT (0.5mg/ml) 1 hour before the addition of IFNγ (5 ng/ml) plus IL-1β (10ng/ml). 24 hours later, islets were dissociated into single cellsuspensions and double-stained for FACS analysis with anti-CD45-APC andanti-MHCII-PE, or isotype control antibodies. Left, Mean±SEM percentchange from control (CT) unstimulated islets. *P<0.05 betweenAAT-treated and untreated islets. Right, Representative FACS analysis;Shaded area, AAT-treated islets. Open area, stimulated islets. Eventsare gated for CD45+.

AAT Modifies Islet Response to Proinflammatory Mediators

Various islet responses to IL-1β/IFNγ were examined in vitro. Isletsexposed to IL-IL-1β/IFNγ for 72 hours produce nitric oxide (NO) in aconcentration-dependent manner and exhibit NO-dependent loss ofviability. As shown in FIGS. 4A and B, in the presence of AAT, less NOwas produced and greater islet viability was obtained. The production ofMIP-1α was decreased in the presence of AAT, particularly whenstimulated by low concentrations of IL-1β/IFNγ (FIG. 4C). Notably, TNFαlevel in supernatants was markedly diminished by AAT (FIG. 4D). Insulininduction was inhibited by IL-1β/IFNγ, but was intact in the presence ofIL-1β/IFNγ plus AAT (FIG. 4E). To test the effect of AAT on islets invivo, STZ toxicity was evaluated. AAT (2 mg) was administered one daybefore, on the same day and a day after STZ injection.Immunohistochemistry of pancreata with anti-insulin antibodies at 48hours after STZ injection reveals more insulin-producing cells in isletsof AAT- than ALB-treated mice (26.3%±2.6 and 12.8%±2.3 insulin-producingcells per islet, respectively, FIG. 4 f). White cell content of freshlyisolated islets was evaluated by FACS analysis. Islets contain CD45+cells (FIG. 4G) that are also positive for the monocytic/granulocyticmarkers GR1 and F4/80 (data not shown). This cell population respondedto AAT with decreased surface MHC class II (FIG. 4H).

Example 5

FIG. 5A-5D illustrates the effect of AAT on TNF-α. (A) Islets fromC57BL/6 mice were cultured (100 islets/well in triplicate) in thepresence of AAT (0.5 mg/ml) or TACE inhibitor (10 mM) 1 hour beforestimulation by IFNγ (5 ng/ml) plus IL-1β (10 ng/ml). Left, mean±SEMchange in TNFα in supernatants after 72 hours of incubation. Right,mean±SEM fold change in membrane TNFα on islet cells after 5 hours ofincubation, according to FACS analysis. ***P<0.001 compared control (CT)levels in the absence of AAT. (B) Representative FACS analysis ofmembrane TNFα on stimulated islet cells in the absence (open area) orpresence (shaded area) of AAT. Events are gated for CD45+. (C)Streptozotocin-induced hyperglycemia. C57BL/6 mice were injected i.p.with saline (n=3), AAT (5 mg, n=3) or TNFα (1 mg/kg, n=3) oradministered p.o. with TACE inhibitor (TACEi, 60 mg/kg, n=6) one daybefore injection of STZ (225 mg/kg, i.p.). Subsequently, AAT and TNFαwere injected daily; TACE inhibitor was administered twice a day. At 48hours, mean±SEM glucose levels are compared to those of normallittermates (n=3). *P<0.05, **P<0.01 compared to saline-treated,STZ-injected mice.

AAT Inhibits Release of Membrane TNFα

Proteolytic cleavage of membrane TNFα releases soluble TNFα fromactivated cells by the action of TNFα-converting-enzyme (TACE). Theinventors examined the levels of membrane TNFα on stimulated islets inthe presence of AAT. The effect of AAT was compared to that of a TACEinhibitor. Both AAT and TACE inhibitor decreased TNFα levels insupernatants of islets exposed to IL-1β/IFNγ (FIG. 5A, left). Underthese conditions, membrane TNFα accumulated on the cell surface of CD45+islet cells (FIG. 5A, right).

To assess the possibility that islet protection occurs via inhibition ofrelease of membrane TNFα in vivo, TACE inhibitor, p75 TNF receptor (TNFBP) or AAT were introduced to mice prior to STZ injection. Although allmice developed hyperglycemia after day 4, the progression of β-celltoxicity was significantly affected by treatments. As shown in FIG. 5C,the effect of STZ at 48 hours was decreased in the presence of AAT (adecrease of 23.2%±2.3 in fasting glucose levels compared to STZ/salineinjected mice). The effect of TACE inhibitor and p75 TNF receptor wasnot as profound. Similarly, TACE inhibitor prolonged islet graftsurvival to a lesser extent than AAT (preliminary data not shown).

Splenocytes that were harvested 48 hours after ThG injection producedTNFα in culture (FIG. 5D). AAT administered prior to thioglycolatedecreased TNFα release from cultured splenocytes. A similar trend wasfound with IFNγ (data not shown), signifying that the response to ThGhad effects that extend beyond the peritoneal compartment and thatpretreatment with AAT reduced these effects.

Example 6

FIG. 6A-6D illustrates the effect of AAT on Islet allografttransplantation. 6A illustrates the time course study aftertransplantation of islet cells. This example indicates that treated micemaintain normoglycemia over a 60 day period (n=4), after the AAT therapywas withdrawn. After withdraw of the therapy, the normoglycemia lastedanother 20 days. 6A illustrates the glucose follow-up. Positive insulinstaining in a day-85 treated islet graft was also demonstrated (data notshown). 6B illustrates an immune infiltrate found outside the graftarea. 6C illustrates an increase in the presence of CD4+ and acomparative decrease in monocytes and neutrophils. It was also shownthat massive vascularization was evident inside the graft (data notshown). It has been observed that long-lasting accepted islet grafts canbe spared from an immune alloresponse even after therapy removal,whether the therapy had evoked an immune tolerance specific for thestrain of donor islets was evaluated. For this, grafts were explanted bynephrectomy and the now-hyperglycemic original recipients werere-transplanted with either the same strain of islets as before (n=2),or a 3^(rd) strain which they had never encountered before (n=2). Inaccordance with established strain specific immune tolerance, miceaccepted grafts from original donors, but had acutely rejected3^(rd)-strain grafts (6D); the same donor (left) and a 3^(rd) donorre-graft (right).

Example 7

FIG. 7A-7E illustrates the production of AAT by islet cell andreflection of islet graft survival. 7A illustrates a time corseexpression of mouse AAT mRNA after cytokine production (IL-1β and IFNγ)(left) and at 8 hours (right). To demonstrate the relevance ofendogenous alpha-1-antitrypsin in physiological conditions, the issue ofislet injury during pancreatitis was addressed. In mouse model of acutepancreatitis, isolated islets of pancreata that are inflamed expressinducible alpha-1-antitrypsin. 7B illustrates an example of islet injuryduring pancreatitis; the histology of normal islets (top left), thehistology of islets of an inflamed pancreas (top right) and expressionof mouse AAT in islets obtained from the pancreata in an acutepancreatitis model (bottom). Alpha-1-antitrypsin levels duringpancreatitis (caerulein model for acute pancreatitis). Top, histology ofan islet in a normal pancreas (left) and an islet in an inflamedpancreas (right), representative of n=3. Bottom, expression of mousealpha-1-antitrypsin in islets obtained from pancreata in acutepancreatitis model. Treatment of mice with exogenous alpha-1-antitrypsinresulted in down-regulation of endogenous alpha-1-antitrypsinexpression, as well as decrease in serum TNFα levels (not shown).

To demonstrate the relevance of endogenous alpha-1-antitrypsin in islettransplantation, islet allografts from untreated transplanted mice ondays 1 through 7 after transplantation (n=3) were excised. These wereexamined for alpha-1-antitrypsin expression and reveal a pattern whichmay fit inflammation phase (days 1-3) followed by loss of islet mass(days 4-7). 7C illustrates an example of samples of islet allograftstaken post grafting and percent change in AAT mRNA levels were alsoassessed. Total RNA was extracted and mRNA for alpha-1-antitrypsinevaluated by RT-PCR.

Islet protection from cytokine injury was examined using endogenousalpha-1-antitrypsin by introducing oncostatin M, a member of IL-6 familythat induces alpha-1-antitrypsin expression in islets without causingislet death. After 4 days that human islets were incubated withoncostatin M, for the purpose of accumulation of sufficientalpha-1-antitrypsin, islets were added the β-cell-toxic combination ofIL-1β/IFNγ. Pretreated islets that had excess alpha-1-antitrypsin wereprotected from injury, supporting the concept that islet-derivedalpha-1-antitrypsin may participate in islet protection duringinflammation. 7D illustrates an example of islet protection fromcytokine injury with endogenous AAT by introducing oncostatin M (aninterleukin 6 (IL-6) family member) that induces AAT expression inislets, oncostatin M and AAT levels (top left); nitric oxide andviability levels assessed (top right). Bottom, human islets exposed tooncostatin M for 4 days produce enough alpha-1-antitrypsin to diminishthe effects of IL-1β/IFNγ added for an additional 48 hours.

Example 8

In one exemplary study, alpha-1-antitrypsin on human islets wasexamined. FIG. 8A-8D illustrates the effect of AAT on human islets. Theproduction of nitric oxide (8A), TNF-α production (8B) IL-6 (8C) andIL-8 (8D) was examined. 100 human islets per well were seeded intriplicates and added alpha-1-antitrypsin (AAT) 2 hours before stimuli.Supernatants were assayed 72 hours later. 3A, nitric oxide; 3B, TNFα;3C, IL-6; 3D, IL-8. Results are mean±SEM and are representative ofseparate islet isolations from three human donors.

Methods

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition 1989, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; Animal Cell Culture,R. I. Freshney, ed., 1986).

Mice.

C57BL/6 and DBA/2 females were purchased from Jackson Laboratories.

Induction of Hyperglycemia by Streptozotocin, Islet Isolation and IsletTransplantation.

In one exemplary method, 5-6 weeks old C57BL/6 mice were treatedintraperitoneally (i.p.) with 225 mg/kg Streptozotocin (STZ) (Sigma).Mice with established hyperglycemia were used at least 5 days after STZadministration. Islets were isolated from DBA/2 mice on day oftransplantation, or 24 hours before in vitro assays, by enzymaticdigestion of pancreata, by means known in the art, with minormodifications. Briefly, mice were anesthetized with i.p. ketamine (50mg/kg, Vedco Inc.) and xylazine (10 mg/kg, Vedco Inc.). Each pancreaswas inflated with 3.5 ml cold collagenase (1 mg/ml, type XI, Sigma),excised and immersed for 40 minutes at 37° C. in water bath. Pancreatawere gently vortexed and filtered through 500-micron metal sieve. Thepellet was washed twice in cold HBSS containing 0.5% BSA (Sigma) andreconstituted in RPMI-1640 (Cellgro, Mediatech) supplemented with 10%FCS (Cellgro), 50 IU/ml Penicillin (Cellgro) and 50 μg/ml streptomycin(Cellgro). Islets were collected on a 100-micron nylon cell strainer (BDFalcon), released into a petri dish by rinsing with HBSS (Cellgro,Mediatech) and 0.5% BSA (Sigma) and hand picked under astereomicroscope. For transplantation, 450 islets were thoroughly washedfrom residual FCS in HBSS and 0.5% BSA and mounted on 0.2 ml tip forimmediate transplantation. For in vitro assays islets were left toincubate for 24 hours at 37° C. Islet transplantation was performed intothe left renal subcapsular space. Recipient mice were anesthetized, asdescribed above. An abdominal wall incision was made over the leftkidney. Islets were released into the subcapsular space through apuncture and the opening was sealed by means known in the art. Bloodglucose follow-up was performed 3 times a week from end-tail blood dropusing glucosticks (Roche). (Nanji, S. A. & Shapiro, A. M. Islettransplantation in patients with diabetes mellitus: choice ofimmunosuppression. BioDrugs 18, 315-28 (2004).)

Development of Anti-Human-AAT Antibodies in Mice.

In another exemplary method, in order to evoke specific antibodyproduction against human AAT, mice were injected i.p. with 10 mg humanAAT per 20-gram mouse for four times in intervals of 1 week. Mice wereused in experiments 2 months after last administration. Antibodyproduction was evaluated before transplantation experiments were carriedout.

In one example, assaying for anti-human-AAT antibody levels wasperformed as described in the art. Briefly, mouse sera were kept at −70°C. until assayed for anti-human-AAT levels. Plates were coated withhuman AAT or albumin (2 μg/ml) in PBS at 4° C. overnight, then washedand blocked for 1 hour at 25° C. as described. Negative control serumwas used in addition to test serum. Bound anti-AAT antibody usingstandard TMB substrate solution was measured (Sigma).

Cells.

NIH-3T3 cell line (e.g. ATCC) were cultured. On day of peritonealinoculation, 1×10⁷ cells were freshly collected by trypsinization andwashed with cold PBS. Pellet was resuspended in 1 ml cold PBS forimmediate injection.

Infiltration Experiments.

Peritoneal infiltration was elicited by i.p. injection of 1 mlautoclaved thioglycolate (3% w/v, Sigma) or allogeneic cells (NIH-3T3),together with 0.1 ml saline, human albumin, human AAT or oxidized AAT.Peritoneal lavage was performed at 24 and 48 hours (thioglycolate) or ondays 1-5 (allogeneic cells). For lavage, mice were anesthetized byisoflurane inhalation and injected immediately with 5.5 ml cold PBScontaining 5% FCS and 5 U/ml heparin into the peritoneal cavity. Aftermassaging the abdomen, peritoneal fluid was recovered. Red blood cellswere lysed (RBC lysing buffer, BD PharMingen) and cell counts wereperformed with a hemocytometer. Cells were then isolated. Cells (about1×10⁶/polypropylene vial) were incubated with FcγRIII/II receptor blockantibodies (Table I) for 10 min. Cells were then divided into two groupsand incubated with mAbs for leukocytes and either CD3/NK cells orneutrophil/monocytes/macrophages (Table I) for 30 min. Cells were washedand fixed. The number of cells expressing a particular marker wascalculated by multiplying percentages obtained from flow-cytometry bythe concentration of cells in lavage fluid.

TABLE I Rat Anti-Mouse mAbs Used for Flow Cytometry Purpose mAb (1)Specificity (2) Source Blocking 2.4G2 FcγRIII/II BD PharMingenLeukocytes 30-F11 (APC) CD45 (leukocytes) BD PharMingen Macrophages andF4/80 (PE) F4/80 (macrophages/monocytes) eBiosciences NeutrophilsRB6-8C5 (FITC) GR1 (neutrophils/monocytes) BD PharMingen CD3 DX5 (PE)Pan-NK cells Miltenyi Biotec NK cells 17A2 (FITC) CD3 BD PharMingen TNFαMP6-XT22 (PE) Mouse TNFα eBiosciences MHC class II M5/114.15.2 (PE)I-A^(b/d), I-E^(d) BD PharMingen Isotype control Rat IgG1 (PE)eBiosciences

An insulin assay and immunohistochemistry were performed by means knownin the art (Nanji, S. A. & Shapiro, A. M. Islet transplantation inpatients with diabetes mellitus: choice of immunosuppression. BioDrugs18, 315-28 (2004)).

AAT Oxidation by Myeloperoxidase (MPO) System.

In one example, AAT (4 mg/ml) was incubated at 37° C. for 45 minuteswith MPO (1 U/ml, Sigma), H₂O₂ (80 μM, Sigma) and NaCl (2.5 mM) in PBS,pH 7.4, by means known in the art. Reaction was terminated by boilingfor 1 hour followed by filter-centrifugation of the system products. Inthis example, boiling was needed for the inactivation of MPO but thisdid not inactivate AAT (data not shown). Loss of activity of oxidizedAAT was confirmed by elastase activity assay.

Elastase Activity Assay.

In another exemplary method, inhibition of a the serine proteaseelastase was evaluated 30 minutes after co-incubation of AAT or oxidizedAAT with porcine elastase (Sigma) in triplicate, by known methods. Theability of elastase to liberate 4-nitroaniline (A₄₁₀) from SucAla₃-PNAwas determined by kinetic measurement of light absorbance at 410 nm.Activity in the absence of inhibitors was set as 100% at the linearrange of the assay.

Cytokine Assays.

An electrochemiluminescence (ECL) assay as known in the art was used forthe measurement of mouse TNFα and MIP-1α. Briefly, cytokine-specificgoat anti-mouse affinity purified antibodies were labeled with ruthenium(e.g. BioVeris) according to manufacturer's instructions. Biotinylatedpolyclonal anti-mouse antibodies (e.g. R&D Systems) were used. Theamount of TNFα and MIP-1α chemiluminescence was determined using anOrigen Analyzer (BioVeris).

Membrane TNFα.

Membrane TNFα on islet cells was detected by modification of a methodfor the evaluation of membrane TNFα on human PBMC. Briefly, single-cellsuspension of islets was incubated with anti-mTNFα-PE mAb (Table I).Cells were washed with FACS buffer and resuspended in 0.5 ml 2% EM-gradeformaldehyde.

Nitric Oxide Assay.

Nitrite levels in supernatants were determined using Griess reagent(Promega), as previously described (Chan, E. D. & Riches, D. W. Am JPhysiol Cell Physiol 280, C441-50 (2001).

Apoptosis Assay.

The protective effect of AAT on islets may address one of the majorobstacles in islet transplantation today, namely the inadequacy of isletmass and post-isolation islet viability. Freshly isolated human isletsactivate stress signaling pathways and exhibit high rate of apoptosisdue to the process of isolation, necessitating the use of more than oneislet donor per diabetic patient (Nanji, (2004); Abdelli, S. et al.Intracellular stress signaling pathways activated during human isletpreparation and following acute cytokine exposure. Diabetes 53, 2815-23(2004)).

In this example, apoptosis that follows islet isolation is diminishedwhen islets are cultured with AAT (data not shown) and demonstrate thatislets that are cultured with AAT for 24 hours prior to transplantationare able to normalize serum glucose levels of diabetic mice whentransplanted autologously at an otherwise sub-functional mass (data notshown).

AAT Dosage.

Normal human plasma contains 0.8-2.4 mg/ml AAT, with a half life of 5-6days¹. In gene transfer studies in C57BL/6 mice, plasma levels of0.8-1.0 mg/ml were achieved and provided protection from type I diabetesin NOD mice (Song, S. et at Gene Therapy 11, 181-6 (2004)). AATadministered intraperitoneally at 0.3-1.0 mg per mouse protected fromTNFα-induced lethal response, and 0.8 mg AAT protected fromD-galactosamine/LPS induced hepatic injury. Libert, C., et al., JImmunol 157, 5126-9 (1996).

Since AAT levels rise 3- to 4-fold during the acute phase response 1, 2mg per mouse results in plasma levels that do not exceed physiologicallevels.

Statistical Analysis.

Comparisons between groups were analyzed by two-sided t-test or ANOVAfor experiments with more than two subgroups. Results are presented asmean±SEM.

Prolongation of Islet Graft Survival by AAT.

In the present study, administration of clinical grade AAT to micetransplanted with allogeneic islets prolonged graft survival. Inaddition, AAT reduced migration of neutrophils and the subsequentinfiltration of lymphocytes and NK cells in models of peritonitis. AATalso decreased secretion of TNFα and MIP-1α from islets and inhibitedsurface MHC class II expression on CD45+ islet cells in vitro. AAT wasprotective in a model of streptozotocin (STZ)-induced β-cell toxicity.Thus, it appears that AAT monotherapy targets several aspects of anactivated inflammatory immune system, culminating in prolongation ofislet allograft survival.

Effect of AAT on Cell Infiltration.

AAT diminished neutrophil migration into the peritoneum of mice injectedwith either thioglycolate or MHC-incompatible fibroblast cells. Otherstudies demonstrate that AAT inhibits neutrophil infiltration intokidneys during ischemia/reperfusion injury and into lungs followingintratracheal administration of silica. In the present study AATdecreased islet production of MIP-1α and TNFα, resulting in isletsdeficient in chemotactic capabilities and therefore less immunogenic.The detrimental effect of neutrophils recruited to islets has beenclearly demonstrated.

The involvement of macrophages in islet destruction is critical; theirpresence precedes insulitis in NOD mice and in prediabetic BB rat, andtheir depletion is protective during islet transplantation in rats.Islets are potent recruiters of macrophages; of the 51 gene productsidentified in freshly isolated human islets by cDNA array, expression ofMCP-1 was found to be high. In mice, blockade of MCP-1 prolongs isletallograft survival when combined with a short subtherapeutic course ofrapamycin. Islet allograft rejection is associated with a steadyincrease in intragraft expression of MCP-2, MCP-5, CCL5, CXCL-10 andCXCL9, and the chemokine receptors CCR2, CCR5, CCR1 and CXCR337.Accordingly, CCR2−/− mice and CXCR3−/− mice exhibit prolongation ofislet allograft survival. In transplant settings, cytokines that areproduced locally, as TNFα and IL-1β, cause damage to proximal cellsindependent of antigen recognition, and complement activation iscritical for graft survival independent of allospecific immunity. Therelevance of macrophages during early events in islet graft rejection isstrengthened by the identification of CD45, F4/80 and Gr1 positive cellsthat express MHC class II in freshly isolated islets. In the presence ofAAT, MHC class II levels were decreased below those ofIL-1β/IFNγ-stimulated and unstimulated islets, supporting the idea thatthe process of islet isolation is sufficient to provoke activation ofinflammatory pathways in islet cells. In light of the involvement ofneutrophils and macrophages in graft rejection, interference with theirfunctions by AAT provides an unusually non-inflammatory environment forthe survival and recovery of engrafted islets.

As shown in the present study and elsewhere intraperitoneal injection ofallogeneic NIH-3T3 cells evokes infiltration of macrophage andneutrophil on days 1-2 and of CD3+ and NK cells on days 4-5. Theintensity of the latter infiltration was decreased by administration ofAAT prior to allogeneic cell-line injection, but not by administrationof AAT on day 3 (data not shown). In transplant settings, earlynon-specific factors contribute to subsequent specific immune response.It is therefore possible that the decrease in CD3+ and NK cellinfiltration in the present study is secondary to the functional failureof the early innate response. However, regardless of AAT treatment,histological examination of islet grafts demonstrated that theinfiltrate evoked by allogeneic islets consists of neutrophils andlymphocytes. Nevertheless, day 7 infiltrate was diminished inAAT-treated recipients, and, according to day 15 insulinimmunohistochemistry, the infiltrate caused less islet destruction.

AAT Inhibits Release of TNFα.

Supernatants of IL-1β/IFNγ-stimulated islets contained strikingly lessTNFα when incubated with AAT (induction of 100.0%±22.0 mean±SEM at 0mg/ml AAT; 10.2%±11.2 at 0.5 mg/ml and 0.8%±0.1 at 1.0 mg/ml). Instimulated human PBMC, AAT was shown to diminish TNFα release withoutaffecting TNFα-mRNA levels. In mice, accordingly, serum TNFα levels aredecreased in LPS-injected AAT-treated mice. Importantly, treatment ofmice with AAT blocks TNFα-mediated LPS-induced, but not TNFα-inducedlethality in mice. In the present study, cultured mouse splenocytesisolated from thioglycolate-injected mice secreted less TNFα, 48 hoursafter injection of AAT.

In the presence of AAT, membrane TNFα accumulated inIL-1β/IFNγ-stimulated CD45+ islet cells. TNFα is released from the cellsurface of macrophages by the action of TNFα converting enzyme (TACE), ametalloproteinase that cleaves membrane TNFα into the soluble form ofTNFα Inhibitors of TACE reduce TNFα release and increase the levels ofmembrane TNFα, as demonstrated by FACS analysis. Although the regulationof TACE activity is unclear, there is evidence to suggest thatextracellular proteases are involved: TACE does not require itscytoplasmic domain for its activation, its activity does not depend onthe amount of TACE on the cell surface, co-expression of TACE andtransmembrane TNFα is not sufficient for processing of TNFα and theenzyme is expressed constitutively in various cells. Serpins, such asserpin PN-I52, are suggested to possess extracellular regulatory effectson various surface proteins.

TACE is likely to be relevant for graft rejection since TACE inhibitordecreased injury parameters in a rat model of post-transplant lunginjury. In addition to a decrease in TNFα levels, the study shows lowerexpression of MCP-1 and ICAM-1, and a reduction in neutrophilinfiltration. Similar findings were obtained with both AAT and a broadspectrum metalloproteinase inhibitor in a model of silica inducedneutrophil influx into lungs. However, TACE inhibitor only partiallyreproduced the protective effect of AAT on islet graft survival(preliminary data). Similarly, AAT protection from STZ-inducedhyperglycemia was only partially reproduced by TACE inhibition and byrecombinant p75-TNF-receptor. Despite the fact that locally secretedTNFα is detrimental to islet graft function, there is, to our knowledge,no report that describes protection of islet grafts by neutralization ofTNFα activity. This distinction between AAT and TACE inhibition supportsthe possibility that AAT affects multiple aspects of the immune system,including not only TNFα release but also events that are downstream toTNFα activities.

In one embodiment, it is contemplated that a composition of the presentinvention may include AAT, an analog thereof, a serine protease, TACEinhibitor (TACEi) or any combination thereof. These compositions may beadministered to a subject having or in need of a transplant and or inneed of immunotolerance therapy.

Transplanted Islets are Stimulated by the Process of Isolation.

The process of islet isolation initiates in the islets an inflammatorycascade of cytokines and chemokines. Thus, isolated islets contain anintrinsic proinflammatory potential that may affect local host immuneresponses. The mechanism of cytokine-induced islet toxicity is believedto involve expression of inducible nitric oxide synthase and subsequentproduction of nitric oxide (NO) by non-β-cells. In the present study,AAT decreased NO production in IL-1β/IFNγ-treated islets. Accordingly,islet viability was increased in a low NO environment, as attained byeither incubation with a low concentration of stimulators (data notshown) or by introduction of AAT. Insulin induction, which is typicallyincomplete in the presence of cytokines, was intact in the presence ofAAT and cytokines. In vivo, AAT protected islets in mice injected withSTZ, as concluded by lower serum glucose levels. The portion of viableβ-cells was visually assessed by insulin immunohistochemistry and wasproportional to the decrease in serum glucose levels. The protection ofAAT was limited to the initial days that follow STZ administration,suggesting that AAT interferes with NO production and immune activationand not with intracellular DNA alkylation. Freshly isolatednon-stimulated CD45+ islet cells expressed MHC class II, which isinvolved in immune responses against islets. The levels of MHC class IIwere elevated in the presence of IL-1β/IFNγ and decreased in thepresence of AAT. Interestingly, MHCII expression was unaffected by thepresence of TACE (TNF alpha converting enzyme) inhibitor (data notshown), confirming that AAT activities extend beyond those of TACEinhibition.

According to the present study, the activities of AAT are directedagainst multiple components of the innate immune system, culminating ina protective effect on islet graft destruction. Islets in particularexhibited a high degree of protection from inflammatory processes in thepresence of AAT. Pretreatment with AAT prior to islet transplantationmay reduce both islet loss and the immunological response against thegraft.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed herein, optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group and that other members of the describedgroups are included but may not be listed.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

All of the COMPOSITIONS and METHODS disclosed and claimed herein may bemade and executed without undue experimentation in light of the presentdisclosure. While the COMPOSITIONS and METHODS have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variation may be applied to the COMPOSITIONS and METHODSand in the steps or in the sequence of steps of the METHODS describedherein without departing from the concept, spirit and scope of theinvention. More specifically, it will be apparent that certain agentswhich are both chemically and physiologically related may be substitutedfor the agents described herein while the same or similar results wouldbe achieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims.

What is claimed:
 1. A method for reducing risk of graft versus hostdisease (GVHD) in a subject, administering to the subject at risk ofdeveloping GVHD, a composition comprising alpha-1 antitrypsin (AAT) orcleavage product thereof, or recombinant molecule thereof, or ketone oraldehyde derivative thereof, and a pharmaceutically acceptableexcipient, and reducing the risk of developing GVHD or reducing effectsof GVHD in the subject.
 2. The method of claim 1, wherein administeringcomprises administering an AAT recombinant molecule comprising a fusionof full-length AAT polypeptide or cleavage product thereof, fused to animmunoglobulin molecule or to a domain of an immunoglobulin molecule orto GST.
 3. The method of claim 1, further comprising administering oneor more anti-rejection agent, anti-inflammatory agent, immunosuppressiveagent, immunomodulatory agent, anti-microbial agent, anti-viral orcombination thereof to the subject.
 4. The method of claim 1, whereinthe GVHD is attributed to a stem cell or bone marrow implant in thesubject.
 5. The method of claim 4, wherein the GVHD is attributed tobone marrow implantation and the subject is administered the compositionfollowing the bone marrow implantation.
 6. The method of claim 2,wherein the immunoglobulin domain comprises an IgG1 constant region. 7.The method of claim 1, wherein the composition is a dose of about 0.1mg/kg to about 100 mg/kg.
 8. A method for treating a subject havinggraft versus host disease (GVHD), said method comprising administeringto the subject a composition comprising alpha-1 antitrypsin (AAA) orrecombinant thereof, and a pharmaceutically acceptable excipient,wherein administering AAT or recombinant thereof to the subject treatsthe subject.
 9. The method of claim 8, wherein the recombinant AAT is anAAT fusion polypeptide comprising full-length alpha1-antitrypsin or acleavage product thereof, fused to an immunoglobulin molecule or to adomain of an immunoglobulin molecule or to GST.
 10. A method forreducing risk of graft versus host disease (GVHD) in a subjectcomprising, administering to the subject at risk of developing GVHD, acomposition consisting essentially of alpha-1 antitrypsin (AAT) orcleavage product thereof, or recombinant molecule thereof, or ketone oraldehyde derivative thereof, and a pharmaceutically acceptableexcipient, to reduce the risk of developing GVHD or reduce effects ofGVHD in the subject.
 11. The method of claim 10, wherein the compositionis administered after organ or non-organ transplantation to reduceincidence of GVHD or effects of GVHD in the subject as compared to acontrol subject not receiving the composition.
 12. The method of claim10, wherein the recombinant AAT is an AAT fusion polypeptide comprisingfull-length alpha1-antitrypsin or a cleavage product thereof, fused toan immunoglobulin molecule or to a domain of an immunoglobulin moleculeor to GST.